Compositions and methods for vas-occlusive contraception

ABSTRACT

Disclosed are methods of delivering an agent to the lumen of the vas deferens under guidance of ultrasound imaging. The methods include vas-occlusive contraception in which the vas deferens is non-surgically isolated and an occlusive substance is percutaneously administered into the lumen of the vas deferens under ultrasound. Also disclosed are methods of reversal of vas-occlusive contraception and methods of delivering an agent to the lumen of the vas deferens. Also disclosed are compositions for use in the methods of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application relies on the disclosure of and claims priority to andthe benefit of the filing date of U.S. Provisional Application No.62/254,381, filed on Nov. 12, 2015 and U.S. Provisional Application No.62/369,807, filed on Aug. 2, 2016, the disclosures of each of which arehereby incorporated by reference herein in their entireties.

BACKGROUND OF THE INVENTION

Field of the Invention

The present invention is generally directed to the field of urology.More particularly, the present invention is directed to the field ofmale contraception. Even more particularly, the present invention isdirected to methods and compositions for male contraception by way ofpercutaneous administration of one or more occlusive substance into thevas deferens. The methods are advantageously performed under theguidance of an imaging modality, such as ultrasound imaging, to ensureplacement of the occlusive substance into the lumen of the vas deferens.Also disclosed are methods of reversal of the male contraception. Alsodisclosed are compositions of occlusive substances that are visible byway of ultrasound imaging and other types of imaging.

Description of Related Art

Vasectomy is a procedure for producing male contraception which involvessevering the vas deferens. Potential complications of vasectomy includebleeding at the site of the surgical procedure, which may cause swellingor bruising; infection at the site of the incision; infection in thescrotum; sperm granuloma; congestive epididymitis; recanalization; andthe inability to reverse the vasectomy. Additionally, a portion ofpatients report pain after the procedure. Possibly the largest deterringfactor of vasectomy, besides the surgical nature of the procedure, isthe difficulty of reversing the vasectomy. The procedure, known asvasovasostomy, is a three to four hour long, expensive microsurgicalprocedure in which the patient is under general anesthesia. Further, avasovasostomy also does not guarantee the man restores his fertility dueto the presence of anti-sperm antibodies that persist in the body afterthe vasovasostomy.

Due to these potential complications and difficulty in reversing theprocedure, alternative procedures for long-lasting male contraceptionhave been explored. One strategy that has been the subject of researchand development is vas-occlusive contraception, which involves injectingor implanting a substance into the vas deferens lumen to occlude thisvessel so that the flow of sperm cells from the epididymis is blocked.Particular examples include RISUG, which involves implantation ofstyrene maleic anhydride, VASALGEL, which involves implantation ofstyrene maleic acid, as well as polyurethane and silicone implants.However, technical barriers for successfully introducing theseprocedures into the male contraceptive armamentarium have beendocumented.

Examples of related efforts include those described in U.S. Pat. Nos.5,488,075; 5,667,767; 6,103,254; 6,858,219; 6,756,031; 8,551,001;8,123,693; 8,613,282; 8,689,792; 8,550,085; 8,434,489; 8,113,2057,975,697; 7,694,683; 7,398,780; 7,073,504; 6,432,116; 6,096,052;8,360,064; 9,034,053; 8,322,341; 8,235,047; 7,918,863; 7,694,683;8,048,086; 9,220,880; 9,034,053; 8,726,906; 8,695,606; 8,336,552;8,324,193; 8,316,854; 8,316,853; 8,052,669; 8,048,101; 8,048,086;8,226,680; 7,789,891; 4,920,982; 8,603,080; 4,269,174; 6,485,426;8,316,854; and 8,551,001; as well as U.S. Patent Application PublicationNos. 2005/0045183; 2015/0068531; 2015/0136144; 2008/0308110;2005/0192616; 2010/0063392; and 2004/0240715. Additional related effortsinclude the following: Reddy, N. M., et al., J. Clin. Ultrasound, 32:394-398 (2004); Abdala, N. et al., Journal of Vascular andInterventional Radiology, 12(8): 979-984 (2001); Zhao, S. C.,Contraception, 41(5):453-459 (1990); Guha, S. K. et al., Contraception,56:4, 245-250 (1997); Chaki, S. P. et al., Contraception, 67(1):73-78(2003); Lohiya, N. K. et al., Contraception, 71(3):214-226 (2005); Liu,X. et al., Contraception, 56(6): 391-394 (1997) (“Liu, X. et al.”);Koul, V. et al., Contraception 58(4):227-31 (1998); Lohiya, N. K., etal., Indian J Med Res 140 (Supplement): 63-72 (2014); Roy, S. et al.,Colloids and Surfaces B: Biointerfaces 69: 77-84 (2009); Zhao, S. C.,International Journal of Andrology, 15:460-464 (1992); Soebadi, D. M.,International Journal of Andrology, 18: 45-52 (1995); Jha, R. K., etal., International Journal of Nanomedicine, 4:55-64 (2009); Middleton,W. D., et al., J Ultrasound Med., 28(7):839-46 (2009); Naughton, C. K.,et al., Journal of Andrology, 25: 545-553 (2004); Waller et al., Basicand Clinical Andrology, 26:6 (2016); and Zambon, J. V., BJUInternational, 86, 699-706 (2000). Yet, despite these efforts, thereremains a need in the art for an improved method of male contraceptionthat can be made available to men in need of a safe, effective, andeasily administered reversible contraceptive.

SUMMARY OF THE INVENTION

According to embodiments, the present invention provides a method whichincludes non-surgically or surgically isolating the vas deferens of asubject, placing an ultrasound probe on or near the vas deferens andadministering ultrasonic energy to image a lumen portion of the vasdeferens. Any aspect of the methods can be performed surgically,non-surgically, or a combination of non-surgically and surgically. Aneedle or catheter or portion thereof can be placed into the lumenportion of the vas deferens, such as percutaneously and/or optionallyunder guidance of ultrasound imaging, and a substance is administeredinto the lumen portion of the vas deferens through the needle orcatheter. In various embodiments, the methods encompass any therapeuticor diagnostic application of administering a substance into the vasdeferens, optionally under ultrasound imaging.

In more particular embodiments, the present invention provides a methodof vas-occlusive contraception involving non-surgical or surgicalisolation of the vas deferens and administration of an occlusivesubstance into the lumen of the vas deferens. In some embodiments, thesubstance administered into the lumen of the vas deferens is a polymer(i.e. vas-occlusive polymer), such as a substance capable of forming apolymer or mass, or a combination of substances capable of forming apolymer or mass when combined. According to embodiments, the entireprocedure, or one or more portions of the procedure, is guided by way ofan imaging modality, such as ultrasound imaging. According toembodiments, the polymer or substance can be administered as a solutionand form an occlusion or mass in the lumen of the vas deferens in situ.Formation of the occlusion or mass can take place in whole or in part inthe lumen. The polymer or substance can be echogenic such that it can bevisualized by way of an imaging modality, such as ultrasound imaging.

In embodiments, the method includes non-surgically or surgicallyisolating the vas deferens and placing an ultrasound probe on or nearthe vas deferens. In some embodiments, the method further includesapplying ultrasonic energy and visually identifying the vas-deferens byway of ultrasound imaging prior to, during, or after administering theocclusive substance. In some embodiments, the method further includesapplying ultrasonic energy and determining an inner (i.e. lumen)diameter, outer diameter, and length of the vas deferens by way ofultrasound imaging prior to, during, or after administering theocclusive substance. In some embodiments, the method further includesapplying ultrasonic energy and identifying the lumen of the vas deferensby way of ultrasound imaging prior to, during, or after administeringthe occlusive substance. In some embodiments, the method furtherincludes applying ultrasonic energy and visually confirming placement ofa needle or catheter or a portion thereof into the lumen of thevas-deferens by way of ultrasound imaging prior to, during, or afteradministering the occlusive substance. In some embodiments, the methodfurther includes applying ultrasonic energy and visually confirmingplacement of the occlusive substance in the lumen of the vas deferens byway of ultrasound imaging. In some embodiments, when the occlusivesubstance is a polymer, such as a substance capable of forming a polymeror mass, or a combination of substances capable of forming a polymer ormass when combined, the method further includes applying ultrasonicenergy and monitoring of polymerization of the echogenic vas-occlusivepolymer in real time by way of ultrasound imaging. In some embodiments,the method further includes determining one or more dimensions of anocclusion formed by the administered substance inside the lumen of thevas deferens by way of ultrasound imaging.

According to embodiments, the non-surgical or surgical isolation of thevas deferens optionally includes use of the “three-finger technique” toisolate the vas deferens close to the scrotal skin. According to otherembodiments, the non-surgical or surgical isolation of the vas deferensalternatively or in addition includes use of a vas-fixation clamp togrip the vas deferens through the skin of the scrotum. In someembodiments, a combination of these techniques is used. Once isolatedand secured beneath the scrotal skin, an occlusive substance such as avas-occlusive polymer (including a substance capable of forming apolymer or mass, or a combination of substances capable of forming apolymer or mass when combined) can be administered into the vas deferensby way of any technique now or later available in the field, such as byway of percutaneous injection or controlled intra-vasal infusion.

According to embodiments, the vas-occlusive polymer, or substance(s)capable of forming a polymer, copolymer, or block copolymer, is firstdissolved in a solvent prior to administration into the vas deferens.Not wishing to be bound by any particular theory, it is believed thatdissipation of the solvent in the aqueous environment of the vasdeferens lumen and/or uptake via epithelial cells causes the polymer topolymerize in situ, or otherwise form as mass thereby eventually formingan occlusion inside the lumen.

According to some embodiments, the occlusive substance such as avas-occlusive polymer is innately echogenic. In some embodiments, thevas-occlusive polymer is echogenic due to the presence of microbubblespresent in the polymer solution. In other embodiments, the vas-occlusivepolymer is echogenic due to other constituents present in the polymersolution.

Embodiments of the invention additionally provide for the use ofultrasonic imaging to confirm placement of the occlusive substance intothe vas deferens lumen, determine location of the occlusion, one or moredimensions of the occlusion such as length and diameter, as well asmonitor the long-term stability of the occlusion in the vas deferens.

According to another embodiment of the invention, a method of reversalof occluding a body lumen, such as a reversal of a vas-occlusivecontraception, is provided. The methods of reversal includenon-surgically or surgically isolating the vas deferens andadministering a solvent into the vas deferens lumen. In embodiments, thesolvent is capable of deteriorating, breaking down, degrading,disintegrating, reversing, dissolving, destroying, removing, dislodging,de-precipitating, liquefying, flushing and/or reducing, in whole orpart, an occlusion or mass, such as a vas-occlusive polymer occlusiondisposed in the lumen of the vas deferens. In some embodiments, themethod includes alternatively or in addition applying ultrasonic energyand visually identifying an echogenic polymer occlusion in the lumen ofthe vas-deferens by way of ultrasound imaging prior to, during, or afteradministering the solvent. In some embodiments, the method furtherincludes alternatively or in addition applying ultrasonic energy andvisually confirming placement of a needle or catheter or a portionthereof into the lumen of the vas-deferens by way of ultrasound imagingprior to, during, or after administering the solvent. In someembodiments, the method further includes alternatively or in additionapplying ultrasonic energy and visually confirming dissolution of theechogenic polymer occlusion disposed in the lumen of the vas deferens byway of ultrasound imaging, for example, during or after administeringthe solvent. In some embodiments, instead of administering a solvent,ultrasonic energy can be applied at an intensity and/or frequencycapable of breaking down the occlusion. For example, the ultrasonicenergy can be applied at an intensity and/or frequency that are capableof lysing microbubbles present in the occlusion, thereby breaking downthe occlusion.

According to another embodiment of the invention, a method of deliveringan agent to the lumen of the vas deferens is provided. The methodincludes formulating or incorporating one or more agents intomicrobubbles of a vas-occlusive polymer solution and/or the solutionitself, non-surgically or surgically isolating the vas deferens of asubject, administering the solution into the lumen of the vas deferens,thereby administering the agent into the lumen, and/or allowing themicrobubbles to lyse thereby releasing the agent into the lumen of thevas deferens. In some embodiments, the method further includes applyingultrasonic energy at a frequency which is capable of lysing themicrobubbles, thereby releasing the agent into the lumen of the vasdeferens. In some embodiments, the method further includes applyingultrasonic energy and visually identifying the lumen of the vas-deferensby way of ultrasound imaging prior to, during, or after administeringthe solution. In some embodiments, the method further includes applyingultrasonic energy and visually confirming placement of a needle orcatheter or a portion thereof into the lumen of the vas-deferens by wayof ultrasound imaging prior to, during, or after administering thesolution. In some embodiments, the method further includes applyingultrasonic energy and visually confirming lysing of the microbubbles inreal time by way of ultrasound imaging.

According to another embodiment, the present invention providescompositions and formulations that are visible by way of ultrasoundimaging and other types of imaging. The compositions and formulationsare designed for use in the methods of the invention. In embodiments,the compositions and formulations include one or more monomers, apolymer or polymers, a copolymer or copolymers, or a block copolymer orblock copolymers. In particular embodiments, different formulations ofethylene vinyl alcohol (EVOH) in dimethyl sulfoxide (DMSO) are designedto have advantageous properties for use in methods of the invention suchas biocompatibility, gelation time, porosity, hardness, viscosity,swelling/fluid absorbance, melting temperature, gelation temperature,sperm motility, sperm viability, degradation, and/or echogenicity.

Specific aspects of embodiments of the invention include Aspect 1, whichare methods of vas-occlusive contraception, comprising: (a) identifyinga vas deferens of a subject in need of contraception; (b) placing anultrasound probe on or near the vas deferens and administeringultrasonic energy to image a lumen portion of the vas deferens, and (c)performing one or more or all of the following steps optionally underguidance of ultrasound imaging: (i) measuring one or more dimensions ofthe lumen portion of the vas deferens; (ii) percutaneously placing aneedle or catheter or portion thereof into the lumen portion of the vasdeferens; (iii) administering a substance into the lumen portion of thevas deferens through the needle or catheter; (iv) confirming formationof an occlusion inside the lumen portion, which occlusion is provided bythe substance administered into the lumen; and/or (v) determining one ormore dimensions of the occlusion inside the lumen portion.

Aspect 2 includes such methods where the substance is a polymer, such asa substance capable of forming a polymer or mass, or a combination ofsubstances capable of forming a polymer or mass when combined, includingfor example one or more monomers, one or more polymers or polymerprecursors, one or more copolymers, or one or more block copolymers, orany combination thereof.

Aspect 3 includes such methods where the polymer is administered as asolution.

Aspect 4 is a method of any of Aspects 1-3, wherein the polymer forms anocclusion in situ by polymerization inside the lumen portion of the vasdeferens.

Aspect 5 is a method of any of Aspects 1-4, wherein the step ofidentifying is performed using a finger technique, such as athree-finger technique. Aspect 6 is a method of any of Aspects 1-5,wherein the step of identifying is further performed using a vas clampto secure the vas deferens to overlying scrotal skin.

Aspect 7 is a method of any of Aspects 1-6, wherein at least the placingand administering steps are performed under guidance of ultrasoundimaging. Aspect 8 is a method of any of Aspects 1-7, wherein at leastthe confirming and determining steps are performed under guidance ofultrasound imaging. Aspect 9 is a method of any of Aspects 1-8, whereinat least the determining step is performed under guidance of ultrasoundimaging. Aspect 10 is a method of any of Aspects 1-9, wherein at leastthe administering and confirming steps are performed under guidance ofultrasound imaging. Aspect 11 is a method of any of Aspects 1-10,wherein at least the placing, administering, confirming, and determiningsteps are performed under guidance of ultrasound imaging.

Aspect 12 is a method of any of Aspects 1-11, wherein the ultrasoundprobe is placed longitudinally to the vas deferens. Aspect 13 is amethod of any of Aspects 1-12, wherein the ultrasound probe is placedaxially to the vas deferens.

Aspect 14 is a method of any of Aspects 1-13, wherein local anesthesiais administered prior to, during or after the placing step.

Aspect 15 is a method of any of Aspects 1-14, wherein the polymersolution comprises a hydrogel. Aspect 16 is a method of any of Aspects1-15, wherein the polymer solution is echogenic.

Aspect 17 is a method of any of Aspects 1-16, wherein the polymersolution is echogenic by way of microbubbles present in the polymersolution. Aspect 18 is a method of any of Aspects 1-17, wherein themicrobubbles are present in the vas-occlusive polymer solution at aconcentration between about 1×10² to about 1×10⁹ microbubbles/ml. Aspect19 is a method of any of Aspects 1-18, wherein the microbubbles have anaverage size in the range from about 1 to about 1,000 μm in diameter.

Aspect 20 is a method of any of Aspects 1-19, wherein the substance isinjected into the lumen portion at a rate of between about 0.10 to about1.0 cc/min.

Aspect 21 is a method of any of Aspects 1-20, wherein the occlusionformed inside the lumen portion comprises a plurality of pores. Aspect22 is a method of any of Aspects 1-21, wherein the pores of theocclusion have a diameter less than about 3 μm. Aspect 23 is a method ofany of Aspects 1-22, wherein the pores of the occlusion have a diameterof between about 0.1 nm to about 1 μm. Aspect 24 is a method of any ofAspects 1-23, wherein the pores of the occlusion have a diameter whichis small enough to restrict passage of sperm cells but large enough topermit passage of fluids.

Aspect 25 is a method of any of Aspects 1-24, wherein the polymersolution has a viscosity in the range of about 1 to about 1000centipoise, or from about 1 to 7 Pa*s, and preferably from about 1-3Pa*s.

Aspect 26 is a method of any of Aspects 1-25, wherein the ultrasonicenergy is administered at a frequency between about 1 and about 20 MHZ.Aspect 27 is a method of any of Aspects 1-26, wherein the ultrasonicenergy is administered at an intensity between about 0.1 to about 1Watts/cm2. Aspect 28 is a method of any of Aspects 1-27, wherein betweenabout 0.1 to about 20 Watts of energy is delivered. Aspect 29 is amethod of any of Aspects 1-28, wherein the ultrasonic energy isadministered in pulsed or continuous mode.

Aspect 30 is a method of any of Aspects 1-29, wherein two substances areinjected together inside a lumen portion of the vas deferens to form anocclusion in situ once they meet. Aspect 31 is a method of any ofAspects 1-30, wherein the two substances are different polymers.

Aspect 32 is a method of any of Aspects 1-31, wherein the polymers areeach present in a solution.

Aspect 33 is a method of any of Aspects 1-32, wherein the vas-occlusivesubstance is a medical device.

Aspect 34 is a method of any of Aspects 1-33, wherein the occlusion iswashed with saline in situ to assist with removal of solvent from thepolymer.

Aspect 35 is a method of any of Aspects 1-34, wherein the substanceincludes a spermicide.

Aspect 36 is a method of any of Aspects 1-35, wherein the substance isadministered into the lumen portion of the vas deferens toward thetestes. Aspect 37 is a method of any of Aspects 1-36, wherein thesubstance is administered into the lumen portion of the vas deferenstoward the prostate.

Aspect 38 is a method of any of Aspects 1-37, wherein the one or moredimensions of the occlusion comprise length, width, and diameter.

Aspect 39 is a method of any of Aspects 1-38, wherein the subject is ananimal. Aspect 40 is a method of any of Aspects 1-39, wherein thesubject is a human.

Aspect 41 is a method of any of Aspects 1-40, wherein the substance isadministered by injection. Aspect 42 is a method of any of Aspects 1-41,wherein the substance is administered by controlled infusion.

Aspect 43 is a method of any of Aspects 1-42, wherein the substance is apolymer and formation of an occlusion due to polymerization of thepolymer is visualized in real time.

Aspect 44 is a method of any of Aspects 1-43, wherein the polymercomprises from about 5 to about 20 weight percent of an ethylene vinylcopolymer, such as ethylene vinyl alcohol (EVOH), dissolved in asolvent, such as an organic solvent, wherein the ethylene vinylcopolymer comprises about 27 to about 48 mole percent of ethylene, andpreferably about 32 mole percent of ethylene. Aspect 45 is a method ofany of Aspects 1-44, wherein the polymer comprises from about 10 toabout 20 weight percent of an ethylene vinyl copolymer. Aspect 46 is amethod of any of Aspects 1-45, wherein the polymer comprises from about15 to about 20 weight percent of an ethylene vinyl copolymer. Aspect 47is a method of any of Aspects 1-46, wherein the polymer comprises fromabout 31 to about 33 mole percent of ethylene. Aspect 48 is a method ofany of Aspects 1-47, wherein the polymer comprises about 32 mole percentof ethylene.

Aspect 49 is a method of any of Aspects 1-48, wherein the solvent isdimethyl sulfoxide.

Aspect 50 is a method of any of Aspects 1-49, wherein the solutionfurther comprises an imaging agent. Aspect 51 is a method of any ofAspects 1-50, wherein the imaging agent comprises one or more ultrasoundcontrast agents. Aspect 52 is a method of any of Aspects 1-51, whereinthe one or more ultrasound contrast agents are microbubbles.

Aspect 53 is a method of reversing vas-occlusive contraception,comprising: identifying a vas deferens of a subject in need of reversalof contraception; placing an ultrasound probe on or near the vasdeferens and administering ultrasonic energy to image a lumen portion ofthe vas deferens, and optionally under guidance of ultrasound imaging,performing one or more or all of the following steps: identifying anocclusion in the vas deferens; percutaneously placing a needle orcatheter or portion thereof into the lumen portion of the vas deferens;administering one or more substance into the lumen portion of the vasdeferens toward the occlusion; and/or confirming removal of theocclusion inside the lumen portion as a result of administering thesubstance.

Aspect 54 is a method of reversing vas-occlusive contraception,comprising: identifying a vas deferens of a subject in need of reversalof contraception; placing at least one ultrasound probe on or near thevas deferens and administering ultrasonic energy to image a lumenportion of the vas deferens, and optionally under guidance of ultrasoundimaging, performing one or more or all of the following steps:identifying an occlusion in the vas deferens; and/or administeringfocused ultrasonic energy at an intensity or frequency capable ofbreaking down, deteriorating, degrading, disintegrating, reversing,dissolving, destroying, removing, dislodging, de-precipitating,liquefying, flushing and/or reducing the occlusion in whole or part.

Aspect 55 is a method of any of Aspects 53-54, wherein the focusedultrasonic energy is capable of lysing microbubbles present in theocclusion.

Aspect 56 is a method of delivering an agent to the lumen of the vasdeferens, the method comprising: identifying the vas deferens of asubject; placing at least one ultrasound probe on or near the vasdeferens; administering one or more solution into the lumen of the vasdeferens, which solution comprises microbubbles incorporating an agent;and/or allowing the microbubbles to dissipate to release the agent inthe lumen of vas deferens.

Aspect 57 is a method of Aspect 56, comprising applying ultrasonicenergy at a frequency which is capable of lysing the microbubblespresent in the solution, thereby releasing the agent into the lumen ofthe vas deferens.

Aspect 58 is a method of any of Aspects 56-57, wherein the agent renderssperm inviable, immotile, and/or infertile. Aspect 59 is a method of anyof Aspects 56-58, wherein the agent is a therapeutic drug,antimicrobial, anti-inflammatory, steroid, hormone, ionic solution,protein, peptide, antibody, nucleic acid, and/or fragment thereof.

Aspect 60 is a method of any of Aspects 56-59, wherein the ultrasoundprobe is a sector, linear, or convex transducer.

Aspect 61 is a method of administering a substance, comprising:identifying a vas deferens of a subject; placing an ultrasound probe onor near the vas deferens and administering ultrasonic energy to image alumen portion of the vas deferens; and optionally under guidance ofultrasound imaging performing one or more or all of the following:percutaneously placing a needle or catheter or portion thereof into thelumen portion of the vas deferens; and/or administering a substance intothe lumen portion of the vas deferens through the needle or catheter.

Aspect 62 is a method of Aspect 61, wherein the substance comprises adiagnostic agent. Aspect 63 is a method of Aspect 61 or 62, wherein thesubstance comprises an imaging agent. Aspect 64 is a method of any ofAspects 61-63, wherein the imaging agent is detectable by way ofradiofrequency, photoacoustic, infrared, or ultrasonic energy. Aspect 65is a method of any of Aspects 61-64, wherein the imaging agent is anultrasound contrast agent. Aspect 66 is a method of any of Aspects61-65, wherein the ultrasound contrast agent comprises microbubbles.Aspect 67 is a method of any of Aspects 61-66, wherein the imaging agentis a dye. Aspect 68 is a method of any of Aspects 61-67, wherein thesubstance comprises a therapeutic agent. Aspect 69 is a method of any ofAspects 61-68, wherein the therapeutic agent provides contraception.Aspect 70 is a method of any of Aspects 61-69, wherein the therapeuticagent is an occlusive substance.

Aspect 71 is a method of any of Aspects 61-70, wherein the occlusivesubstance is a polymer. Aspect 72 is a method of any of Aspects 61-71,wherein the polymer is administered as a solution.

Aspect 73 is a method of any of Aspects 61-72, wherein the therapeuticagent comprises a sclerotic agent. Aspect 74 is a method of any ofAspects 61-73, wherein the therapeutic agent enhances fertility of thesubject. Aspect 75 is a method of any of Aspects 61-74, wherein thetherapeutic agent reduces motility, viability, or fertility of spermcells.

Aspect 76 is a composition comprising from about 5 to about 20 weightpercent of an ethylene vinyl copolymer, such as ethylene vinyl alcohol(EVOH), dissolved in a solvent, such as an organic solvent, wherein theethylene vinyl copolymer comprises about 27 to about 48 mole percent ofethylene, and preferably about 32 mole percent of ethylene.

Any of the compositions disclosed in this specification can be used inany of the methods, uses, or systems disclosed herein, including themethods, uses, and systems for example of Aspects 1-73, 99-103, 108,111-119, 122-126, 128-139, and 159-179.

Aspect 77 is the composition of Aspect 76, wherein the compositioncomprises from about 10 to about 20 weight percent of an ethylene vinylcopolymer. Aspect 78 is the composition of Aspect 76 or 77, wherein thecomposition comprises from about 15 to about 20 weight percent of anethylene vinyl copolymer. Aspect 79 is the composition of any of Aspects76-78, wherein the composition comprises from about 31 to about 33 molepercent of ethylene. Aspect 80 is any of the compositions of Aspects76-79, wherein the composition comprises about 32 mole percent ofethylene.

Aspect 81 is any of the compositions of Aspects 76-80, wherein thesolvent is dimethyl sulfoxide.

Aspect 82 is any of the compositions of Aspects 76-81 comprising animaging agent. Aspect 83 is any of the compositions of Aspects 76-82,wherein the imaging agent comprises one or more ultrasound contrastagents. Aspect 84 is any of the compositions of Aspects 76-83, whereinthe one or more ultrasound contrast agents are microbubbles. Aspect 85is any of the compositions of Aspects 76-84, wherein the microbubblesare present in the composition at a concentration between about 1×10² toabout 1×10⁹ microbubbles/ml. Aspect 86 is any of the compositions ofAspects 76-85, wherein the microbubbles have an average size in therange from about 1 to about 1,000 μm in diameter.

Aspect 87 is any of the compositions of Aspects 76-86 comprising anagent that decreases the fertility, motility, or viability of spermcells. Aspect 88 is any of the compositions of Aspects 76-87, whereinthe agent is a small molecule, protein, antibody, peptide, nucleic acid,or fragment thereof. Aspect 89 is any of the compositions of Aspects76-88, wherein the agent is nonoxynol-9, octoxynol-9, benzalkoniumchloride, or chlorhexidine.

Aspect 90 is any of the compositions of Aspects 76-89, wherein themicrobubbles comprise a gas or a plurality of gases. Aspect 91 is any ofthe compositions of Aspects 76-90, wherein the gas or plurality of gasesis air. Aspect 92 is any of the compositions of Aspects 76-91, whereinthe gas comprises nitrogen, argon, or perfluorocarbon. Aspect 93 is anyof the compositions of Aspects 76-92, wherein the microbubbles have ashell comprising a polymer, a lipid, a protein, a surfactant, amonosaccharide, a polysaccharide, or glass.

Aspect 94 is any of the compositions of Aspects 76-93, wherein thepolymer comprises one or more of natural or synthetic monomers, polymersor copolymers, biocompatible monomers, polymers or copolymers,polystyrene, neoprene, polyetherether 10 ketone (PEEK), carbonreinforced PEEK, polyphenylene, PEKK, PAEK, polyphenylsulphone,polysulphone, PET, polyurethane, polyethylene, low-density polyethylene(LDPE), linear low-density polyethylene (LLDPE), high-densitypolyethylene (HDPE), polypropylene, polyetherketoneetherketoneketone(PEKEKK), nylon, TEFLON® TFE, polyethylene terephthalate (PETE), TEFLON®FEP, TEFLON® PFA, and/or polymethylpentene (PMP) styrene maleicanhydride, styrene maleic acid, polyurethane, silicone, polymethylmethacrylate, polyacrylonitrile, poly (carbonate-urethane), poly(vinylacetate), nitrocellulose, cellulose acetate, urethane,urethane/carbonate, polylactic acid, polyacrylamide (PAAM), poly(N-isopropylacrylamine) (PNIPAM), poly (vinylmethylether), poly(ethylene oxide), poly (ethyl (hydroxyethyl) cellulose), poly(2-ethyloxazoline), polylactide (PLA), polyglycolide (PGA),poly(lactide-co-glycolide) PLGA, poly(e-caprolactone), polydiaoxanone,polyanhydride, trimethylene carbonate, poly(β-hydroxybutyrate),poly(g-ethyl glutamate), poly(DTH-iminocarbonate), poly(bisphenol Aiminocarbonate), poly(orthoester) (POE), polycyanoacrylate (PCA),polyphosphazene, polyethyleneoxide (PEO), polyethylglycol (PEG),polyacrylacid (PAA), polyacrylonitrile (PAN), polyvinylacrylate (PVA),polyvinylpyrrolidone (PVP), polyglycolic lactic acid (PGLA),poly(2-hydroxypropyl methacrylamide) (pHPMAm), poly(vinyl alcohol)(PVOH), PEG diacrylate (PEGDA), poly(hydroxyethyl methacrylate) (pHEMA),N-isopropylacrylamide (NIPA), poly(vinyl alcohol) poly(acrylic acid)(PVOH-PAA), collagen, silk, fibrin, gelatin, hyaluron, cellulose,chitin, dextran, casein, albumin, ovalbumin, heparin sulfate, starch,agar, heparin, alginate, fibronectin, fibrin, keratin, pectin, elastin,ethylene vinyl acetate, polyethylene oxide, PEG or any of itsderivatives, PLLA, PDMS, PIPA, PEVA, PILA, PEG styrene, Teflon RFE,FLPE, Teflon FEP, methyl palmitate, NIPA, polycarbonate,polyethersulfone, polycaprolactone, polymethyl methacrylate,polyisobutylene, nitrocellulose, medical grade silicone, celluloseacetate, cellulose acetate butyrate, polyacrylonitrile, PLCL, and/orchitosan.

Aspect 95 is any of the compositions of Aspects 76-94, wherein themicrobubbles comprise an agent that decreases the fertility, motility,or viability of sperm cells. Aspect 96 is any of the compositions ofAspects 76-95, wherein the agent is cross-linked to the microbubbles.Aspect 97 is any of the compositions of Aspects 76-96, wherein the agentenclosed within a shell of the microbubbles. Aspect 98 is any of thecompositions of Aspects 76-97, wherein the agent is a small molecule,protein, antibody, peptide, nucleic acid, or fragment thereof.

Aspect 99 is a method of occluding a lumen comprising: identifying alumen; administering ultrasonic energy to image the lumen, andperforming one or more or all of the following steps optionally underguidance of ultrasound imaging: measuring one or more dimensions of thelumen; percutaneously placing a needle or catheter or portion thereofinto the lumen; administering a substance into the lumen through theneedle or catheter; confirming formation of an occlusion inside thelumen; and/or determining one or more dimensions of the occlusion insidethe lumen.

Aspect 100 is the method of Aspect 99, wherein the lumen is a lumen of abody chosen from one or more of vas deferens, fallopian tube, aneurysm,blood vessel, ducts, tumors, and organs. Aspect 101 is the method ofAspect 100, wherein the substance administered into the lumen is any oneor more of the compositions disclosed in this specification, includingin particular any one or more of the compositions of Aspects 76-98.

Aspect 102 is use of any one or more of the compositions of Aspects76-98 for the manufacture of a medicament for the treatment of fertilityor infertility of a male or female human or animal subject. Aspect 103is use according to Aspect 102, wherein the medicament is avas-occlusive contraceptive.

Aspect 104 is a composition of any one or more of the compositions ofAspects 76-98 for use in the treatment of fertility or infertility of amale or female human or animal subject. Aspect 105 is the composition ofAspect 104, wherein the composition is a vas-occlusive contraceptive.

Aspect 106 is the composition of any one or more of the compositions ofAspects 76-98 for use as a medicament. Aspect 107 is the composition ofAspect 106, wherein the medicament is a vas-occlusive contraceptive.

Aspect 108 is a system for treating infertility or fertility of a maleor female human or animal subject, the system comprising: one or moreultrasound imaging apparatus; one or more of any of the compositionsdisclosed in this specification, including any one or more of those ofAspects 76-98, and optionally provided in a syringe or catheter.

Aspect 109 is any one or more of the compositions disclosed in thisspecification, including those of Aspects 76-98, wherein the compositionhas a viscosity in the range of about 1 to about 1000 centipoise, orfrom about 1 to 7 Pa*s, and preferably from about 1-3 Pa*s. Aspect 110is the composition of Aspect 109, wherein the composition exhibits aNewtonian fluid behavior rather than shear thinning.

Aspect 111 is the method of any method disclosed in this specification,including any of the methods disclosed in Aspects 1, 61, or 99, whereinthe substance or solution comprises a polymer and DMSO and the substanceor solution is allowed to precipitate, polymerize, or otherwise forminto a hydrogel and the DMSO leaks out, followed by absorption of theDMSO into a lining of the vas deferens.

Aspect 112 is the method of any Aspect disclosed in this specification,including for example Aspect 15, wherein the hydrogel is formulated toswell no more than 80% based on volume of the hydrogel. Aspect 113 isthe method of any Aspect disclosed in this specification, including forexample Aspect 15, wherein the hydrogel is formulated to swell only somuch that the hydrogel does not completely erode epithelial cells orbasement membrane of the vas deferens, such as formulated to swell butnot completely erode epithelial cells or basement membrane of the vasdeferens.

Aspect 114 the method of any Aspect disclosed in this specification,including for example Aspect 15, wherein the hydrogel in the vasdeferens has a plug length, which is a length capable of blocking spermfrom traversing through the hydrogel, for example the minimal lengththat achieves the highest efficacy qualified by azoospermia or lack ofsperm that are able to traverse the hydrogel.

Aspect 115 is the method of Aspect 114, wherein the plug length isdependent on polymer composition, monomer ratio, molecular weight,concentration in solvent, such as in organic solvent, injection volume,and/or injection speed of the polymer solution, and/or diameter of theinner lumen of the vas deferens.

Aspect 116 is the method of Aspect 114 or 115, wherein the plug lengthis longer than the average length of the smooth muscle fibers in the vasdeferens, such that when the vas deferens constricts and contractsduring peristalsis, the hydrogel resists being dislodged, or does notget dislodged, or is prevented from being dislodged.

Aspect 117 is the method of any of Aspects 114-116, wherein the pluglength is determined based on lumen diameter, injection volume, and acoefficient (Beta), which is the volume of the plug/injection volume ofthe polymer-DMSO solution and is a function of the polymer formulation,such as the composition, molecular weight, monomer ratio, and/orconcentration.

Aspect 118 is the method of any of Aspects 114-117, wherein Beta is >1for hydrogels such that the hydrogel takes up more volume than theinjection volume, such as a Beta of >2 or >3, such that an injectionvolume of 100 uL does not exceed 3-4 cm plug length. Aspect 119 is themethod of any of Aspects 114-118, wherein the greater the Beta, thelesser the plug length.

Aspect 120 is a composition of any Aspect or composition disclosed inthis specification, including those of Aspects 76-98, which isnon-biodegradable in a human or animal body or begins to degrade in ahuman or animal body 1-3 years after implantation, or 2-5 years afterimplantation, or 3-10 years after implantation, or longer. Aspect 121 isthe composition of Aspect 120, wherein the life span of the compositionis days, weeks, months, or years, and is preferably permanent.

Aspect 122 is the method of Aspect 101, wherein the substance is allowedto form, polymerize, or precipitate into a hydrogel occlusion in the vasdeferens and DMSO leaks out from the substance, followed by absorptionof the DMSO into a lining of the vas deferens.

Aspect 123 is the method of Aspect 122, wherein the occlusion is capableof causing sperm that come in contact with the occlusion inviable orimmotile.

Aspect 124 is the composition of any of Aspects 76-98, wherein thecomposition is capable of causing sperm that come in contact with thecomposition inviable or immotile.

Aspect 125 is any method disclosed in this specification, includingthose of Aspects 1, 53, 54, 56, 61, or 99, which is capable of beingperformed and completed in no more than 10 minutes, such as no more than5 minutes, such as from 3-5 minutes.

Aspect 126 is any method disclosed in this specification, includingthose of Aspects 1, 53, 54, 56, 61, or 99, wherein an occlusion isformed and the occlusion is washed with saline in situ to assist withremoval of solvent from the polymer.

Aspect 127 is a polymer formed from any of the compositions of Aspects76-98, wherein the polymer has a weight average molecular weight (M_(w))or number-average molecular weight (M_(n)) ranging from about 1,000 to1,000,000 Daltons.

Aspect 128 is the method of Aspect 1, wherein the identifying stepincludes non-surgically identifying the vas deferens.

Aspect 129 is any method disclosed in Aspects 1-128, wherein theidentifying step includes surgically identifying the vas deferens.Aspect 130 is any method disclosed in Aspects 1-129, wherein theidentifying step includes non-surgically and surgically identifying thevas deferens.

Aspect 131 is any method disclosed in Aspects 1-130 comprisingadministering a vasodilator prior to, during, or after the identifyingstep.

Aspect 132 is any method disclosed in Aspects 1-131, wherein thesubstance comprises microbubbles. Aspect 133 is any method disclosed inAspects 1-132, wherein the microbubbles have a shell comprising apolymer, a lipid, a protein, a surfactant, a monosaccharide, apolysaccharide, or glass. Aspect 134 is any method disclosed in Aspects1-133, wherein the microbubbles are formed by way of double syringeagitation.

Aspect 135 is any method disclosed in Aspects 1-134, wherein the one ormore substance is a therapeutic agent capable of producing a therapeuticeffect upon male urological function. Aspect 136 is any method disclosedin Aspects 1-135, wherein the substance is one or more substance chosenfrom a contraceptive, an occlusive device, a therapeutic agent, andimaging agent, and echogenic agent, microbubbles, spermicide,antimicrobials, vasodilators, and/or a composition or device forincreasing or decreasing motility, viability, and/or fertility of spermor ova.

Aspect 137 is any method disclosed in Aspects 1-136, wherein thesolution comprises a therapeutic drug, antimicrobial, anti-inflammatory,steroid, hormone, ionic solution, protein, peptide, antibody, nucleicacid, and/or fragment thereof.

Aspect 138 is any method disclosed in Aspects 1-137, wherein thesubstance is sterile or is subject to one or more sterilization method.Aspect 139 is any method disclosed in Aspects 1-138, wherein thesubstance is sterilized by one or more of using a DMSO compatible 0.22micron filter, dry heat, autoclave, ethylene oxide, gamma, and/or e-beambased sterilization method.

Aspect 140 is a composition comprising: about 5 to about 20 weightpercent of an ethylene vinyl copolymer; wherein the ethylene vinylcopolymer is dissolved in a solvent; wherein the ethylene vinylcopolymer comprises about 27 to about 48 mole percent of ethylene.

Aspect 141 is the composition of Aspect 140 comprising about 10 to about20 weight percent of the ethylene vinyl copolymer. Aspect 142 is thecomposition of Aspect 140 or Aspect 141 comprising about 31 to about 33mole percent of ethylene.

Aspect 143 is the composition of any of Aspects 140-142, which has aviscosity in the range of about 1 to 7 Pa*s. Aspect 144 is thecomposition of any of Aspects 140-143, which has a viscosity in therange of about 1-3 Pa*s.

Aspect 145 is the composition of any of Aspects 140-144, wherein theethylene vinyl copolymer is ethylene vinyl alcohol (EVOH).

Aspect 146 is the composition of any of Aspects 140-145, wherein thesolvent is an organic solvent.

Aspect 147 is the composition of any of Aspects 140-146 comprising oneor more ultrasound contrast agents.

Aspect 148 is the composition of any of Aspects 140-147 comprising oneor more microbubbles. Aspect 149 is the composition of any of Aspects140-148, wherein the microbubbles are present in the composition at aconcentration between about 1×10² to about 1×10⁹ microbubbles/ml. Aspect150 is the composition of any of Aspects 140-149, wherein themicrobubbles have an average size in the range from about 1 to about1,000 μm in diameter.

Aspect 151 is the composition of any of Aspects 140-150, wherein themicrobubbles have a shell encompassing a gas or a plurality of gaseschosen from one or more of air, nitrogen, argon, or perfluorocarbon.

Aspect 152 is the composition of any of Aspects 140-151, wherein theshell of the microbubbles comprises a polymer, a lipid, a protein, asurfactant, a monosaccharide, a polysaccharide, or glass.

Aspect 153 is the composition of any of Aspects 140-152, wherein theshell of the microbubbles comprises a polymer comprising one or more ofnatural or synthetic monomers, polymers, copolymers or block copolymers,biocompatible monomers, polymers, copolymers or block copolymers,polystyrene, neoprene, polyetherether 10 ketone (PEEK), carbonreinforced PEEK, polyphenylene, PEKK, PAEK, polyphenylsulphone,polysulphone, PET, polyurethane, polyethylene, low-density polyethylene(LDPE), linear low-density polyethylene (LLDPE), high-densitypolyethylene (HDPE), polypropylene, polyetherketoneetherketoneketone(PEKEKK), nylon, TEFLON® TFE, polyethylene terephthalate (PETE), TEFLON®FEP, TEFLON® PFA, and/or polymethylpentene (PMP) styrene maleicanhydride, styrene maleic acid, polyurethane, silicone, polymethylmethacrylate, polyacrylonitrile, poly (carbonate-urethane), poly(vinylacetate), nitrocellulose, cellulose acetate, urethane,urethane/carbonate, polylactic acid, polyacrylamide (PAAM), poly(N-isopropylacrylamine) (PNIPAM), poly (vinylmethylether), poly(ethylene oxide), poly (ethyl (hydroxyethyl) cellulose), poly(2-ethyloxazoline), polylactide (PLA), polyglycolide (PGA),poly(lactide-co-glycolide) PLGA, poly(e-caprolactone), polydiaoxanone,polyanhydride, trimethylene carbonate, poly(β-hydroxybutyrate),poly(g-ethyl glutamate), poly(DTH-iminocarbonate), poly(bisphenol Aiminocarbonate), poly(orthoester) (POE), polycyanoacrylate (PCA),polyphosphazene, polyethyleneoxide (PEO), polyethylglycol (PEG),polyacrylacid (PAA), polyacrylonitrile (PAN), polyvinylacrylate (PVA),polyvinylpyrrolidone (PVP), polyglycolic lactic acid (PGLA),poly(2-hydroxypropyl methacrylamide) (pHPMAm), poly(vinyl alcohol)(PVOH), PEG diacrylate (PEGDA), poly(hydroxyethyl methacrylate) (pHEMA),N-isopropylacrylamide (NIPA), poly(vinyl alcohol) poly(acrylic acid)(PVOH-PAA), collagen, silk, fibrin, gelatin, hyaluron, cellulose,chitin, dextran, casein, albumin, ovalbumin, heparin sulfate, starch,agar, heparin, alginate, fibronectin, fibrin, keratin, pectin, elastin,ethylene vinyl acetate, polyethylene oxide, PEG or any of itsderivatives, PLLA, PDMS, PIPA, PEVA, PILA, PEG styrene, Teflon RFE,FLPE, Teflon FEP, methyl palmitate, NIPA, polycarbonate,polyethersulfone, polycaprolactone, polymethyl methacrylate,polyisobutylene, nitrocellulose, medical grade silicone, celluloseacetate, cellulose acetate butyrate, polyacrylonitrile, PLCL, and/orchitosan.

Aspect 154 is the composition of any of Aspects 140-153 comprising anagent that decreases the fertility, motility, or viability of spermcells. Aspect 155 is the composition of any of Aspects 140-154, whereinthe agent is one or more of a small molecule, protein, antibody,peptide, nucleic acid, or fragment thereof, or nonoxynol-9, octoxynol-9,benzalkonium chloride, or chlorhexidine.

Aspect 156 is the composition of any of Aspects 140-155, which is ahydrogel formulated such that when the hydrogel is exposed to water thehydrogel swells no more than 80% by volume.

Aspect 157 is the composition of any of Aspects 140-156, which iscapable of polymerizing into a polymer and wherein the polymer isnon-biodegradable in a human or animal body or begins to degrade in ahuman or animal body 1-3 years after implantation, or 2-5 years afterimplantation, or 3-10 years after implantation, or longer.

Aspect 158 is a composition comprising: ethylene vinyl alcohol (EVOH)dissolved in dimethyl sulfoxide (DMSO); wherein the EVOH comprises about5-20 wt % of ethylene vinyl copolymer; wherein the ethylene vinylcopolymer comprises about 27-48 mole percent ethylene; wherein the EVOHis capable of polymerizing into a polymer comprising a plurality ofpores having an average pore size in the range of about 0.1 nm to about3 μm.

Aspect 159 is a method of occluding a body lumen comprising: imaging abody lumen using ultrasound; and percutaneously placing a needle orcatheter or portion thereof into the lumen; administering thecomposition of Aspect 1 into the lumen through the needle or catheter;and allowing the composition to form an occlusion in the lumen.

Aspect 160 is a method of occluding a body lumen comprising: imaging ananimal or human body lumen to view an image of the body lumen;administering a substance into the body lumen; and polymerizing thesubstance or forming a mass from the substance in the body lumen.

Aspect 161 is the method of Aspect 160, wherein the imaging step isperformed prior to, during, and/or after the administering orpolymerizing or forming a mass steps.

Aspect 162 is the method of Aspect 160 or 161, wherein as a result ofthe polymerizing or forming a mass step an occlusion is formed in situin the body lumen.

Aspect 163 is the method of any of Aspects 160-162, wherein theocclusion is echogenic.

Aspect 164 is the method of any of Aspects 160-163, wherein microbubblesare present in the substance and the microbubbles have an average sizein the range from about 1 to about 1,000 μm in diameter.

Aspect 165 is the method of any of Aspects 160-164, wherein theocclusion comprises a plurality of pores having an average pore size inthe range of about 0.1 nm to about 3 μm.

Aspect 166 is the method of any of Aspects 160-165, wherein, wherein theocclusion comprises a plurality of pores sized to restrict passage ofsperm cells but permit passage of fluids through the pores.

Aspect 167 is the method of any of Aspects 160-166, wherein thesubstance is a composition having a viscosity in the range of about 1 to7 Pa*s.

Aspect 168 is the method of any of Aspects 160-167, wherein themicrobubbles have a shell comprising a polymer, a lipid, a protein, asurfactant, a monosaccharide, a polysaccharide, or glass.

Aspect 169 is the method of any of Aspects 160-168, wherein thesubstance includes one or more of a contraceptive, an occlusive device,a therapeutic agent, and imaging agent, and echogenic agent,microbubbles, spermicide, antimicrobials, vasodilators, and/or acomposition or device for increasing or decreasing motility, viability,and/or fertility of sperm or ova.

Aspect 170 is the method of any of Aspects 160-169, wherein the polymercomprises one or more of natural or synthetic monomers, polymers,copolymers or block copolymers, biocompatible monomers, polymers,copolymers or block copolymers, polystyrene, neoprene, polyetherether 10ketone (PEEK), carbon reinforced PEEK, polyphenylene, PEKK, PAEK,polyphenylsulphone, polysulphone, PET, polyurethane, polyethylene,low-density polyethylene (LDPE), linear low-density polyethylene(LLDPE), high-density polyethylene (HDPE), polypropylene,polyetherketoneetherketoneketone (PEKEKK), nylon, TEFLON® TFE,polyethylene terephthalate (PETE), TEFLON® FEP, TEFLON® PFA, and/orpolymethylpentene (PMP) styrene maleic anhydride, styrene maleic acid,polyurethane, silicone, polymethyl methacrylate, polyacrylonitrile, poly(carbonate-urethane), poly (vinylacetate), nitrocellulose, celluloseacetate, urethane, urethane/carbonate, polylactic acid, polyacrylamide(PAAM), poly (N-isopropylacrylamine) (PNIPAM), poly (vinylmethylether),poly (ethylene oxide), poly (ethyl (hydroxyethyl) cellulose),poly(2-ethyl oxazoline), polylactide (PLA), polyglycolide (PGA),poly(lactide-co-glycolide) PLGA, poly(e-caprolactone), polydiaoxanone,polyanhydride, trimethylene carbonate, poly(β-hydroxybutyrate),poly(g-ethyl glutamate), poly(DTH-iminocarbonate), poly(bisphenol Aiminocarbonate), poly(orthoester) (POE), polycyanoacrylate (PCA),polyphosphazene, polyethyleneoxide (PEO), polyethylglycol (PEG),polyacrylacid (PAA), polyacrylonitrile (PAN), polyvinylacrylate (PVA),polyvinylpyrrolidone (PVP), polyglycolic lactic acid (PGLA),poly(2-hydroxypropyl methacrylamide) (pHPMAm), poly(vinyl alcohol)(PVOH), PEG diacrylate (PEGDA), poly(hydroxyethyl methacrylate) (pHEMA),N-isopropylacrylamide (NIPA), poly(vinyl alcohol) poly(acrylic acid)(PVOH-PAA), collagen, silk, fibrin, gelatin, hyaluron, cellulose,chitin, dextran, casein, albumin, ovalbumin, heparin sulfate, starch,agar, heparin, alginate, fibronectin, fibrin, keratin, pectin, elastin,ethylene vinyl acetate, polyethylene oxide, PEG or any of itsderivatives, PLLA, PDMS, PIPA, PEVA, PILA, PEG styrene, Teflon RFE,FLPE, Teflon FEP, methyl palmitate, NIPA, polycarbonate,polyethersulfone, polycaprolactone, polymethyl methacrylate,polyisobutylene, nitrocellulose, medical grade silicone, celluloseacetate, cellulose acetate butyrate, polyacrylonitrile, PLCL, and/orchitosan.

Aspect 171 is the method of any of Aspects 160-170, wherein thesubstance is a composition comprising from about 5 to about 20 weightpercent of an ethylene vinyl copolymer dissolved in a solvent, whereinthe ethylene vinyl copolymer comprises about 27 to about 48 mole percentof ethylene.

Aspect 172 is the method of any of Aspects 160-171, wherein thecomposition comprises from about 10 to about 20 weight percent of anethylene vinyl copolymer and from about 31 to about 33 mole percent ofethylene.

Aspect 173 is the method of any of Aspects 160-172, wherein thecomposition comprises dimethyl sulfoxide (DMSO) as the solvent andethylene vinyl alcohol (EVOH) as the ethylene vinyl copolymer.

Aspect 174 is the method of any of Aspects 160-173, wherein thesubstance comprises an imaging agent.

Aspect 175 is the method of any of Aspects 160-174, wherein the bodylumen is chosen from one or more of a vas deferens, fallopian tube,aneurysm, blood vessel, ducts, tumors, and organs.

Aspect 176 is a method of removing an occlusion disposed in a bodylumen, comprising: imaging a body lumen and an occlusion disposedtherein; performing one or more or all of the following: administering asubstance into the body lumen and allowing the substance to deteriorate,break down, degrade, disintegrate, reverse, dissolve, destroy, remove,dislodge, de-precipitate, liquefy, flush and/or reduce the occlusion inwhole or part; and/or administering ultrasonic energy to the occlusionat an intensity and/or frequency capable of deteriorating, breakingdown, degrading, disintegrating, reversing, dissolving, destroying,removing, dislodging, de-precipitating, liquefying, flushing and/orreducing the occlusion in whole or part; confirming deterioration of theocclusion.

Aspect 177 is the method of Aspect 176, wherein the body lumen is chosenfrom one or more of a vas deferens, fallopian tube, aneurysm, bloodvessel, ducts, tumors, and organs.

Aspect 178 is a method of occluding a body lumen comprising: imaging abody lumen with ultrasound; injecting a composition comprising ethylenevinyl alcohol (EVOH) and dimethyl sulfoxide (DMSO) into the body lumen;and polymerizing the composition or forming a mass from the compositionin situ in the body lumen.

Aspect 179 is the method of Aspect 178, wherein the body lumen is chosenfrom one or more of a vas deferens, fallopian tube, aneurysm, bloodvessel, ducts, tumors, and organs.

Additional embodiments and aspects of the invention will be apparent inthe foregoing detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings illustrate certain aspects of embodiments ofthe present invention, and should not be used to limit the invention.Together with the written description the drawings serve to explaincertain principles of the invention.

FIG. 1A is diagram illustrating a method of vas-occlusive contraceptionaccording to an embodiment of the invention.

FIG. 1B is a diagram showing the result of the method of FIG. 1A.

FIGS. 2, 4-7, and 10 are longitudinal ultrasound images of the vasdeferens in a human male.

FIGS. 3, 8, and 9 are axial ultrasound images of the vas deferens in ahuman male.

FIG. 11 is a longitudinal ultrasound image of a synthetic vas deferens.

FIG. 12 is a longitudinal ultrasound image of a synthetic vas deferensfilled with an EVOH 32-15% polymer gel implant.

FIG. 13A is an axial ultrasound image of the synthetic vas deferens ofFIG. 11.

FIG. 13B is a graph of the grayscale values of FIG. 13A.

FIG. 14A is an axial ultrasound image of the synthetic vas deferensfilled with an EVOH 32-15% polymer gel implant of FIG. 12.

FIG. 14B is a graph of the grayscale values of FIG. 14A.

FIG. 15A is a diagram representing the chemical structure of styrenemaleic acid (SMA).

FIG. 15B is a diagram representing the chemical structure of ethylenevinyl alcohol (EVOH).

FIG. 16 is a microscopic image of polystyrene microbubbles.

FIGS. 17A and 17B are graphs showing lateral (FIG. 17A) and axial (FIG.17B) resolutions of various echogenic EVOH gel images.

FIG. 18 is a graph showing the results of a cell viability (MTT) assayof EVOH, SMA, and control samples.

FIG. 19 is a table showing the results of statistical analysis (Tukeytesting) of the results of FIG. 18.

FIG. 20 is graph showing the results of a cell viability (MTT) assay ofselect polymer candidates.

FIG. 21 is a table showing the results of statistical analysis (Tukeytesting) of the results of FIG. 20 comparing EVOH 27, EVOH 32, and EVOH38.

FIG. 22 is a table showing the results of statistical analysis (Tukeytesting) of the results of FIG. 21 comparing EVOH at different weightpercentages (5%, 10%, 15%, and 20%).

FIG. 23 is a table showing the results of a cytotoxicity assay (MEMelution) for select EVOH polymer candidates.

FIG. 24 is a photograph showing results of injection of a polymer in thevas deferens in a rodent model.

FIGS. 25A-25D are histopathology images of the vas deferens of 12 weekold, Sprague-Dawley male rats which received injections of a SMA polymersolution.

FIGS. 26A-25C are histology images of the vas deferens which representthe change in the luminal diameter and content of the vas deferens afterinjection of an EVOH polymer solution.

FIGS. 27A-27D are histology images which show the resting diameter ofthe epithelial lamina propria.

FIG. 28 is a table showing the relationship between inner diameter(dilated) and injection volume and for various targeted occlusion sizesfor human vas-occlusive contraception.

FIG. 29 is a table showing results of Brunauer-Emmett-Teller analysisfor select polymer candidates.

FIG. 30A shows a scanning electron microscopy image for the EVOH 32-10%of FIG. 29.

FIG. 30B shows a scanning electron microscopy image for the EVOH 32-15%of FIG. 29

FIG. 30C shows a scanning electron microscopy image for the EVOH 32-20%of FIG. 29

FIG. 31 is a table showing the hardness and elastic modulus of selectpolymer candidates.

FIG. 32 is a graph of the results of FIG. 31.

FIG. 33 is a graph showing the results of a sperm motility assay ofselect polymer candidates.

FIG. 34A is a table showing the results of statistical analysis (initialANOVA) of the results of FIG. 33.

FIG. 34B is table showing the results of statistical analysis (Tukeytesting) of the results of FIG. 33.

FIG. 35 is a graph showing the results of a sperm viability assay ofselect polymer candidates.

FIG. 36A is a table showing the results of statistical analysis (initialANOVA) of the results of FIG. 35.

FIG. 36B is table showing the results of statistical analysis (Tukeytesting) of the results of FIG. 36.

FIG. 37 is a graph showing viscosity as a function of shear rate forselect polymer candidates.

FIG. 38 is a graph showing the effect of ethylene mol % on the viscosityof EVOH as a function of shear rate using the controlled shear ratetest.

FIGS. 39-41 are graphs showing melting temperatures and glass transitiontemperatures for EVOH 32-10%, EVOH 32-15%, and EVOH 38-15%,respectively.

FIG. 42 is a graph showing mass percent swelling for select polymercandidates.

FIG. 43 is a graph showing percent fluid absorbance for select polymercandidates.

FIG. 44 is a graph showing the results of a degradation study for selectEVOH polymer candidates. Mass is plotted on the y axis and date isplotted in the x axis.

FIG. 45 is a graph showing the results of a degradation study for selectSMA polymer candidates. Mass is plotted on the y axis and date isplotted in the x axis.

FIG. 46 is an array of ultrasound images showing axial cross-sections ofselect polymer gel candidates.

FIGS. 47A-47C are ultrasound images showing three of the EVOH 32 gels inan expanded view.

FIG. 48 is a graph showing an average pixel brightness and standarddeviation of ultrasound images for select polymer candidates.

DETAILED DESCRIPTION OF VARIOUS EMBODIMENTS OF THE INVENTION

Reference will now be made in detail to various exemplary embodiments ofthe invention. It is to be understood that the following discussion ofexemplary embodiments is not intended as a limitation on the invention.Rather, the following discussion is provided to give the reader a moredetailed understanding of certain aspects and features of the invention.

As used herein, a “subject” is an animal. Such animals include mammals,including companion animals such as a dog or cat, farm animals such as ahorse or cow, laboratory animals such as a mouse or rat, as well asnon-human primates and humans.

As used herein, a “subject in need of contraception” is a patient,animal, mammal, or human, who will benefit from the method of thisinvention.

The term “biocompatible,” as used herein, refers to a material that doesnot elicit a substantial detrimental response in the host.

The term “about,” as used herein, means approximately, in the region of,roughly, or around. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 10%. In one aspect, the term “about” meansplus or minus 20% of the numerical value of the number with which it isbeing used. Therefore, about 50% means in the range of 40%-60%.Numerical ranges recited herein by endpoints include all numbers andfractions subsumed within that range (e.g. 1 to 5 includes 1, 1.5, 2,2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbersand fractions thereof are presumed to be modified by the term “about.”

A “fragment” or “segment” is a portion of an amino acid sequence,comprising at least one amino acid, or a portion of a nucleic acidsequence comprising at least one nucleotide. The terms “fragment” and“segment” are used interchangeably herein.

As used herein, the term “fragment,” as applied to a protein or peptide,can ordinarily be at least about 3-15 amino acids in length, at leastabout 15-25 amino acids, 10 at least about 25-50 amino acids in length,at least about 50-75 amino acids in length, at least about 75-100 aminoacids in length, and greater than 100 amino acids in length.

As used herein, the term “fragment” as applied to a nucleic acid, mayordinarily be at least about 20 nucleotides in length, typically, atleast about 50 nucleotides, more typically, from about 50 to about 100nucleotides, preferably, at least about 100 to about 200 nucleotides,even more preferably, at least about 200 nucleotides to about 300nucleotides, yet even more preferably, at least about 300 to about 350,even more preferably, at least about 350 nucleotides to about 500nucleotides, yet even more preferably, at least about 500 to about 600,even more preferably, at least about 600 nucleotides to about 620nucleotides, yet even more preferably, at least about 620 to about 650,and most preferably, the nucleic acid fragment will be greater thanabout 650 nucleotides in length.

As used herein, the term “nucleic acid” encompasses RNA as well assingle and double-stranded DNA and cDNA. Furthermore, the terms,“nucleic acid,” “DNA,” “RNA” and similar terms also include nucleic acidanalogs, i.e. analogs having other than a phosphodiester backbone. Forexample, the so-called “peptide nucleic acids,” which are known in theart and have peptide bonds instead of phosphodiester bonds in thebackbone, are considered within the scope of the present invention. By“nucleic acid” is meant any nucleic acid, whether composed ofdeoxyribonucleosides or ribonucleosides, and whether composed ofphosphodiester linkages or modified linkages such as phosphotriester,phosphoramidate, siloxane, carbonate, carboxymethylester, acetamidate,carbamate, thioether, bridged phosphoramidate, bridged methylenephosphonate, bridged phosphoramidate, bridged phosphoramidate, bridgedmethylene phosphonate, phosphorothioate, methylphosphonate,phosphorodithioate, bridged phosphorothioate or sulfone linkages, andcombinations of such linkages. The term nucleic acid also specificallyincludes nucleic acids composed of bases other than the fivebiologically occurring bases (adenine, guanine, thymine, cytosine, anduracil). Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction. Thedirection of 5′ to 3′ addition of nucleotides to nascent RNA transcriptsis referred to as the transcription direction. The DNA strand having thesame sequence as an mRNA is referred to as the “coding strand”;sequences on the DNA strand which are located 5′ to a reference point onthe DNA are referred to as “upstream sequences”; sequences on the DNAstrand which are 3′ to a reference point on the DNA are referred to as“downstream sequences.”

The term “peptide” typically refers to short polypeptides.

A “polynucleotide” means a single strand or parallel and anti-parallelstrands of a nucleic acid. Thus, a polynucleotide may be either asingle-stranded or a double-stranded nucleic acid.

“Polypeptide” refers to a polymer composed of amino acid residues,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof.

As used herein, an “effective amount” or “therapeutically effectiveamount” means an amount sufficient to produce a selected effect, such asalleviating symptoms of a disease or disorder. In the context ofadministering compounds in the form of a combination, such as multiplecompounds, the amount of each compound, when administered in combinationwith another compound(s), may be different from when that compound isadministered alone. Thus, an effective amount of a combination ofcompounds refers collectively to the combination as a whole, althoughthe actual amounts of each compound may vary. The term “more effective”means that the selected effect is alleviated to a greater extent by onetreatment relative to the second treatment to which it is beingcompared.

As used herein, a “non-surgical” (or “non-surgically”) aspect of aprocedure is performed without surgery. “Non-surgical isolation” refersto any non-surgical technique which identifies an anatomical partthrough the intact skin or connective tissues such that it is positionedunderneath the skin or connective tissue. Thus, “non-surgical isolation”does not involve the use of a scalpel to incise the skin or the use ofsutures to close the skin after incision.

As used herein, the word “VASINTOMY™” refers to any embodiment of thecontraceptive methods described in this disclosure.

As used in this specification, ethylene vinyl alcohol (EVOH)compositions are referred to as “EVOH X-Y %”, where X is the ethylenecontent (mole percentage) of EVOH and Y % is the weight percentage ofEVOH in the composition. For example, EVOH 32-10% indicates that theEVOH in the composition has an ethylene content of 32%, a vinyl alcoholcontent of 68%, and EVOH represents 10% of the composition by weight.

In embodiments, the present invention relates generally to methods ofadministration of a substance into any lumen in the body, such as thelumen of the vas deferens, optionally under guidance of ultrasoundthrough non-surgical or surgical routes of administration. In apreferred embodiment, the substance is administered percutaneously.Further, the present invention contemplates any therapeutic ordiagnostic aspect of the methods of the invention that can beappreciated by a skilled artisan.

For example, one embodiment provides a method which includesnon-surgically or surgically isolating a vas deferens of a subject,placing an ultrasound probe on or near the vas deferens andadministering ultrasonic energy to image a lumen portion of the vasdeferens, and optionally under guidance of ultrasound imagingpercutaneously placing a needle or catheter or portion thereof into thelumen portion of the vas deferens, and administering a substance intothe lumen portion of the vas deferens through the needle or catheter.

The substance can include any substance that has the potential toproduce a therapeutic effect upon male urological function, or serve asa diagnostic of urological function. For example, the substance can beor include a diagnostic agent such as an imaging agent.

The imaging agent can be detectable by way of any form of energy used indiagnostic imaging, such as, for example, by way of radiofrequency,photoacoustic, infrared, or ultrasonic energy. For example, the imagingagent can be or include an ultrasound contrast agent such asmicrobubbles or can be or include a dye. Additionally, the substance canbe or include a therapeutic agent. In one particular embodiment, theagent is therapeutic in the sense that it produces a desirablecontraceptive effect. For example, the therapeutic agent can be anocclusive substance (otherwise known herein as a vas-occlusivesubstance) that exerts a desired contraceptive effect by blocking thetransport of sperm cells through the vas deferens lumen. The therapeuticagent can include any non-occlusive substance, such as small molecules,antibodies, peptides, proteins, nucleic acids, and the like.

In one particular embodiment, the therapeutic agent is a polymer such asthose described further herein, and the polymer is administered as asolution and results in polymerization in situ to form an occlusion inthe vas deferens. However, the present invention contemplates any agentthat can exert a contraceptive mechanism of action in the vas deferens.Additional contraceptive agents are contemplated, including scleroticagents, which cause vessels to shrink by producing endothelial damage.Alternatively, the therapeutic agent can target sperm cells and exert acontraceptive effect through reduced motility, viability, or fertilityof the sperm cells.

In alternative embodiments, the therapeutic agent can be an agent thatenhances fertility, such as an agent that has an effect of increasingsperm transport, or enhances motility, viability, or fertility of spermcells, if such is the therapeutic goal. In one embodiment, thetherapeutic agent may be an antibiotic. These are merely illustrativeexamples of the different applications of the invention and are notintended to limit the scope. This invention contemplates that any agentcan be administered in the methods of administration of substances intothe vas deferens, such that the method covers any therapeutic ordiagnostic urological application.

While the invention generally relates to methods of administration of asubstance into the lumen of the vas deferens optionally under guidanceof ultrasound through non-surgical or surgical routes of administration,more particular embodiments concerning contraceptive implementations ofthe methods will be discussed. Particularly advantageous embodiments ofthe present invention for which there is a critical need for includemethods of vas-occlusive contraception that are minimally invasive,highly accurate, reproducible, safe, long-lasting, and easy to implementin an outpatient setting. According to embodiments, these advantagesstem from the use of ultrasound imaging throughout at least a portion ofthe procedure.

The inventors' advantageous implementation of ultrasound to guide theplacement of a vas-occlusive substance into the lumen of the vasdeferens has not been previously established in the contraceptive arts.Even further, the use of a non-surgical or surgical method for isolatingthe vas deferens and placement of a contraceptive in the lumen by way ofpercutaneous injection or controlled intra-vasal infusion is asignificant improvement over previous vas-occlusive procedures indevelopment, which generally require an incision to isolate the vasdeferens. Moreover, the inventive procedure can be performed by aphysician using local anesthesia in an outpatient setting very rapidly,in as little as five minutes, thus minimizing any discomfort to thesubject. Moreover, the inventive procedure requires minimal training toperform, such that it can be performed by any medical practitionerfamiliar with ultrasound imaging. Even further, while the inventiveprocedure provides long-lasting contraception, it is easily reversibleby way of a similar procedure. These advantages of the invention, onlysome of which are discussed herein, will be further apparent as variousembodiments and features of the invention are described.

One embodiment of the invention includes a method of vas-occlusivecontraception. The method can include non-surgically or surgicallyisolating a vas deferens of a subject in need of contraception, placingan ultrasound probe on or near the vas deferens and administeringultrasonic energy to image the lumen of the vas deferens, and performingat least one of the following steps optionally under guidance ofultrasound imaging, such as (a) determining one or more dimensions ofthe lumen of the vas deferens (b) percutaneously placing a needle orcatheter or portion thereof into the lumen of the vas deferens, (c)administering a substance into the lumen of the vas deferens, (d)confirming formation of an occlusion inside the lumen as a result ofadministering the substance, and (e) determining one or more dimensionsof the occlusion inside the lumen portion.

According to embodiments, the non-surgical or surgical isolation of thevas deferens includes use of a vas-fixation clamp to grip the vasdeferens through the skin of the scrotum, and/or subcutaneous isolationthrough the physician's fingers. For example, the vas deferens can beisolated through the use of the “three-finger technique,” in which thenon-dominant hand is used to manipulate the vas into a subcutaneousposition (see Stockton D M et al. “No-scalpel vasectomy: a technique forfamily physicians.” Am Fam Physician. 1992; 46:1153-67; Clenney T L,“Vasectomy Techniques,” Am Fam Physician. 1999; 60(1):137-146).Alternatively or in addition, a vas-fixation clamp can be used to securethe vas deferens in a subcutaneous position. Further, the vas clamp cancontain an additional component that guides the needle into the lumen ofthe vas deferens. Additionally, the needle may include a ball-and-socketjoint which allows for easier manipulation of the needle.

In embodiments, the substance is any substance which forms an occlusioninside the vas deferens when administered into the lumen. The substancecan be solid, semi-solid, or liquid. Conceivably, any substance that canbe administered into the vas deferens lumen and forms a plug orocclusion can be used. Preferably, the substance is biocompatible. Thesubstance can be organic or inorganic. The substance can form anocclusion in situ through a phase state change, such as from liquid tosolid. The substance can form an occlusion by expansion in situ. Thesubstance can also be a medical device, such as a nanoparticle,stent-like device, electro-spun mesh, or a nanobot. In embodiments, thesubstance is selectively porous such that is allows fluids to pass whileblocking the passage of sperm cells. In some embodiments, the substanceis a composition. In some embodiments, the substance is a polymer. Insome embodiments, more than one substance is administered. In someembodiments, the substance is selectively toxic to sperm cells but nottoxic to other cells of the genitourinary tissues. The selectivetoxicity can result in decreased motility, viability, or fertility ofthe sperm cells.

According to embodiments, a vas-occlusive polymer can then beadministered into the vas deferens of both sides by way of percutaneousinjection or controlled intra-vasal infusion into the vas, optionallyunder the guidance of ultrasound imaging. For example, the vas-occlusivepolymer can be dissolved in a solvent, preloaded in a syringe, andinjected into the lumen of the vas deferens by way of a needle orcatheter. The needle or catheter is chosen to be of a size that fitsinside the lumen of the vas deferens. For example, one publication hasestimated the lumen of the vas deferens in humans to vary from 0.25 to0.55 mm in diameter, while having an external diameter of between 2 and4 mm (see E. S. Hafez, P. Kenemans, “Atlas of Human Reproduction: ByScanning Electron Microscopy,” 1982, MTP Press, Hingham, Mass.). Thus,the inner diameter (lumen) of the vas is only a small portion of itsouter diameter. However, various other studies have shown that the innerdiameter can dilate as large as 1.8 mm (Liu, X. et al.). The outerportion of the vas is made up of layers of smooth muscle. This sizedifferential underscores the need for ultrasound imaging to confirmsuccessful administration of the vas-occlusive substance into the lumenrather than “off-target” administration into the smooth muscle. The sizeof the needle or catheter can be chosen based on the estimated size ofthe vas from the literature, or determined by imaging the dimensions ofthe vas lumen of the subject through ultrasound. In embodiments, thesize of the needle can be between 18 gauge to 34 gauge, depending on theestimated or determined diameter of the vas lumen of the particularspecies that is the target of contraception. In other embodiments, thesize of the needle is between 21 gauge and 31 gauge. In otherembodiments, the size of the needle is at least 23 gauge, such asbetween 23 gauge and 29 gauge. In another aspect, the needle that isused to deliver the injection solution contains bores on the side, whichallow for the solution to be excreted around the needle, in addition tothe bevel.

According to embodiments, the vas-occlusive polymer is first prepared asa solution. The solution can be prepared at a concentration of polymerranging from about 0.1 to about 1.0 g/ml (or higher) of solvent medium.In other embodiments, the vas-occlusive polymer is prepared as asolution at a concentration ranging from about 0.25 to about 0.75 g/ml.In some embodiments, the concentration is about 0.1, 0.15, 0.2, 0.25,0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9,0.95, or 1.0 g/ml. Various solvents for dissolving the above polymersinclude dimethyl sulfoxide (DMSO), or other organic solvents, orsolutions of sodium bicarbonate, and the like. The solvents may alsocontain agents that selectively dissolve or de-precipitate the polymerby breaking the cross-links including, but not limited to, reducingagents. The polymers can be prepared as a solution using these solventsand delivered (e.g. injected) into the lumen of the vas deferens by wayof a needle or catheter. In some embodiments, the polymer gel can bewashed with saline after delivery to fully form the occlusion and helpwith removal of solvents such as DMSO. In some embodiments, twodifferent polymer solutions are prepared. The two different polymersolutions together are capable of forming a hydrogel when they are mixedtogether. For example, the two different polymer solutions can beadministered into the vas deferens and can form a hydrogel inside thelumen once they meet. Examples of such systems include PEG-thiol andPEG-maleamide, which form a cross-linked hydrogel via “Click” reactionupon mixing.

Polymers that are generally useful for forming an occlusion or embolisminside a vessel are known in the art. Non-limiting examples of suchpolymers include styrene maleic anhydride, styrene maleic acid,polyurethane, silicone and those described in International PatentApplication Publication No. WO 2015058169. Additional non-limitingexamples include hydrogels such as such as polymethyl methacrylate,polyacrylonitrile, poly (carbonate-urethane), poly (vinylacetate),nitrocellulose, cellulose acetate, urethane, urethane/carbonate,polylactic acid, polyacrylamide (PAAM), poly (N-isopropylacrylamine)(PNIPAM), poly (vinylmethylether), poly (ethylene oxide), poly (ethyl(hydroxyethyl) cellulose), poly(2-ethyl oxazoline), polylactide (PLA),polyglycolide (PGA), poly(lactide-co-glycolide) PLGA,poly(e-caprolactone), polydiaoxanone, polyanhydride, trimethylenecarbonate, poly(β-hydroxybutyrate), poly(g-ethyl glutamate),poly(DTH-iminocarbonate), poly(bisphenol A iminocarbonate),poly(orthoester) (POE), polycyanoacrylate (PCA), polyphosphazene,polyethyleneoxide (PEO), polyethylglycol (PEG), polyacrylacid (PAA),polyacrylonitrile (PAN), polyvinylacrylate (PVA), polyvinylpyrrolidone(PVP), polyglycolic lactic acid (PGLA), poly(2-hydroxypropylmethacrylamide) (pHPMAm), poly(vinyl alcohol) (PVOH), PEG diacrylate(PEGDA), poly(hydroxyethyl methacrylate) (pHEMA), N-isopropylacrylamide(NIPA), poly(vinyl alcohol) poly(acrylic acid) (PVOH-PAA), or naturalpolymers such as collagen, silk, fibrin, gelatin, hyaluron, cellulose,chitin, dextran, casein, albumin, ovalbumin, heparin sulfate, starch,agar, heparin, alginate, fibronectin, fibrin, keratin, pectin, orelastin. Additional examples include those described in U.S. Pat. No.6,878,384, which discloses that hydrogels can be prepared by forming aliquid reaction mixture that contains a) monomer(s) and/or polymer(s) atleast portion(s) of which are sensitive to environmental changes (e.g.,changes in pH or temperature), b) a crosslinker and c) a polymerizationinitiator. Additional examples of hydrogels include those described inU.S. Patent Application Publication No. 20090053276A1 and U.S. Pat. Nos.6,703,047; 5,612,052; 5,714,159; 6,413,539; 4,804,691; 6,723,781;5,866,554; 6,037,331; 6,514,534; 6,297,337; 6,514,535; and 5,702,717. Inembodiments, the polymers can be prepared as a solution prior toadministration into the lumen of the vas.

Additional non-limiting examples include ethylene vinyl acetate,polyethylene oxide, PEG or any of its derivatives, PLLA, PDMS, PIPA,PEVA, PILA, PEG styrene, PEEK, nylon, Teflon RFE, PEKEKK, FLPE,neoprene, PETE, Teflon FEP, Teflon PFA, PMP, methyl palmitate, NIPA,polycarbonate, polyethersulfone, polycaprolactone, polymethylmethacrylate, polypropylene, polyurethane, polystyrene, polyisobutylene,nitrocellulose, medical grade silicone, cellulose acetate, celluloseacetate butyrate, polyacrylonitrile, PLCL, and/or chitosan.

In some embodiments, the polymer is a copolymer such as a blockcopolymer, an alternating copolymer, a graft copolymer, a randomcopolymer, a periodic copolymer, and a statistical copolymer. In aparticular embodiment, the copolymer is ethylene vinyl alcohol (EVOH).According to another specific embodiment, the copolymer is EVOHdissolved in dimethylsulfoxide (DMSO).

In one embodiment, the EVOH has a weight percent from around 1% toaround 50% of the complete polymer composition in DMSO, such as from 1%to 2%, from 2% to 3%, from 3% to 4%, from 4% to 5%, from 5% to 6%, from6%, to 7%, from 7%, to 8%, from 8% to 9%, from 9% to 10%, and so on. Inanother embodiment, the weight percent of the EVOH is from around 6% toaround 20% of the complete polymer composition, including 7% to around20%, 8% to around 20%, 9% to around 20%, as so on. In anotherembodiment, the weight percent of the EVOH is around 10% to around 20%of the complete polymer composition, including 10%, 11%, 12%, 13%, 14%,15%, 16%, 17%, 18%, 19%, 20%, and any percentage in between. In anotherembodiment, the weight percent of the EVOH is around 10% to around 18%of the complete polymer composition. EVOH begins to reach its saturationpoint at 20 wt %. Further, as the wt % increases, so does the viscositymaking injection of the material difficult. Thus, solutions at a weightpercentage of greater than 20% are less desirable.

In another aspect, the weight percent of EVOH is chosen such that whenthe polymer gel is formed, the pores of the gel are small enough toprevent sperm from traveling through. In one aspect, the EVOH may havean effect on sperm motility, fertility, and viability. In anotheraspect, sperm that interact with EVOH may lose biomarkers,carbohydrates, or proteins that are important for fertilization.

In one embodiment, the ethylene content in the EVOH can be a molar ratio(mole percent) of around 20% to around 60% of the polymer, including20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, and 60%, or any percentage inbetween these values. According to one embodiment, the ethylene contentis a molar ratio of about 27% to about 44% of the polymer. According toanother embodiment, the ethylene content is in the range of from about27% to about 38% of the polymer, including 27%, 28%, 29%, 30%, 31%, 32%,33%, 34%, 35%, 36%, 37%, and 38%. According to another embodiment, theethylene content is in the range of about 31% to about 33% of thepolymer. According to another embodiment, the ethylene content is about32% of the polymer. The ethylene content controls thehydrophilicity/hydrophobicity of the polymer: the lower the ethylenecontent, the higher the vinyl alcohol and therefore, greaterhydrophilicity. These factors can impact how the hydrogel forms inwater, swells, and absorbs water, as well as its viscosity, thermalstability, and durability. In embodiments, the hydrogel swells no morethan 80%, such as no more than 75%, or no more than 70%, or no more than65%, or no more than 60% and so on. Additionally, the percentage ofethylene can impact the injection force required for administration. Forexample, a 27% ethylene-content allows for an easy injection because thepolymer gel does not adhere to the inside of the needle.

According to embodiments, the EVOH polymeric gel may produce a partialor full occlusion when injected into the vas. In one aspect, the EVOHpolymer may block sperm, fluids, molecules such as proteins (e.g.,antibodies), carbohydrates, gases, and/or lipids. In another aspect, theEVOH gel may occlude sperm due to small pore size but may allow fluidsand other molecules to pass. In another aspect, the EVOH has spermicidalproperties causing the sperm that come in contact to become inviable orimmotile.

Embodiments of the invention provide inventive compositions of EVOH foruse in the methods of the invention. By adjusting the molecular weightsand monomer ratios of EVOH, the present inventors have demonstrated thatother polymer properties important for contraception can be tailored.The inventive compositions are designed to optimize various propertiesthat are relevant to the methods of the invention, includingbiocompatibility, gelation time, porosity, hardness, viscosity,swelling/fluid absorbance, melting temperature, gelation temperature,sperm motility, sperm viability, degradation, and/or echogenicity.

Embodiments of the invention include a composition comprising EVOHdissolved in a solvent. Preferably the solvent is DMSO, but thecompositions can include any biocompatible solvent or solvents.Non-limiting examples of the compositions expressed as “EVOH X-Y %”where X is the ethylene content (mole percentage) of EVOH and Y % is theweight percentage of EVOH in the composition include the following: EVOH27-8%, EVOH 27-9%, EVOH 27-10%, EVOH 27-11%, EVOH 27-12%, EVOH 27-13%,EVOH 27-14%, EVOH 27-15%, EVOH 27-16%, EVOH 27-17%, EVOH 27-18%, EVOH27-19%, EVOH 27-20%, EVOH 28-8%, EVOH 28-9%, EVOH 28-10%, EVOH 28-11%,EVOH 28-12%, EVOH 28-13%, EVOH 28-14%, EVOH 28-15%, EVOH 28-16%, EVOH28-17%, EVOH 28-18%, EVOH 28-19%, EVOH 28-20%, EVOH 29-8%, EVOH 29-9%,EVOH 29-10%, EVOH 29-11%, EVOH 29-12%, EVOH 29-13%, EVOH 29-14%, EVOH29-15%, EVOH 29-16%, EVOH 29-17%, EVOH 29-18%, EVOH 29-19%, EVOH 29-20%,EVOH 30-8%, EVOH 30-9%, EVOH 30-11%, EVOH 30-12%, EVOH 30-13%, EVOH30-14%, EVOH 30-15%, EVOH 30-16%, EVOH 30-17%, EVOH 30-18%, EVOH 30-19%,EVOH 30-20%, EVOH 31-8%, EVOH 31-9%, EVOH 31-10%, EVOH 31-11%, EVOH31-12%, EVOH 31-13%, EVOH 31-14%, EVOH 31-15%, EVOH 31-16%, EVOH 31-17%,EVOH 31-18%, EVOH 31-19%, EVOH 31-20%, EVOH 32-8%, EVOH 32-9%, EVOH32-10%, EVOH 32-11%, EVOH 32-12%, EVOH 32-13%, EVOH 32-14%, EVOH 32-15%,EVOH 32-16%, EVOH 32-17%, EVOH 32-18%, EVOH 32-19%, EVOH 32-20, 3 EVOH3-8%, EVOH 33-9%, EVOH 33-10%, EVOH 33-11%, EVOH 33-12%, EVOH 33-13%,EVOH 33-14%, EVOH 33-15%, EVOH 33-16%, EVOH 33-17%, EVOH 33-18%, EVOH33-19%, EVOH 33-20%, EVOH 34-8%, EVOH 34-9%, EVOH 34-10%, EVOH 34-11%,EVOH 34-12%, EVOH 34-13%, EVOH 34-14%, EVOH 34-15%, EVOH 34-16%, EVOH34-17%, EVOH 34-18%, EVOH 34-19%, EVOH 34-20%, EVOH 35-8%, EVOH 35-9%,EVOH 35-10%, EVOH 35-11%, EVOH 35-12%, EVOH 35-13%, EVOH 35-14%, EVOH35-15%, EVOH 35-16%, EVOH 35-17%, EVOH 35-18%, EVOH 35-19%, EVOH 35-20%,EVOH 36-8%, EVOH 36-9%, EVOH 36-10%, EVOH 36-11%, EVOH 36-12%, EVOH36-13%, EVOH 36-14%, EVOH 36-15%, EVOH 36-16%, EVOH 36-17%, EVOH 36-18%,EVOH 36-19%, EVOH 36-20%, EVOH 37-8%, EVOH 37-9%, EVOH 37-10%, EVOH37-11%, EVOH 37-12%, EVOH 37-13%, EVOH 37-14%, EVOH 37-15%, EVOH 37-16%,EVOH 37-17%, EVOH 37-18%, EVOH 37-19%, EVOH 37-20%, EVOH 38-8%, EVOH38-9%, EVOH 38-10%, EVOH 38-11%, EVOH 38-12%, EVOH 38-13%, EVOH 38-14%,EVOH 38-15%, EVOH 38-16%, EVOH 38-17%, EVOH 38-18%, EVOH 38-19%, EVOH38-20%, and so on. However, these compositions are intended to be merelyexemplary. A skilled artisan can appreciate that the present inventionincludes other compositions not explicitly stated, such as those havingan ethylene content or EVOH weight percentage outside of these ranges orbetween values that are explicitly stated.

In the foregoing examples at the end of this disclosure, the presentinventors have developed novel EVOH compositions with respect toethylene content and EVOH concentrations that have optimal propertieswith respect to one or more of biocompatibility, gelation time,porosity, hardness, viscosity, swelling/fluid absorbance, meltingtemperature, gelation temperature, sperm motility, sperm viability,degradation, and echogenicity. The compositions are optimized for use inthe inventive methods. Thus, the present invention encompasses novelEVOH compositions that have been specifically designed for use inultrasound-guided vas-occlusive contraception.

In embodiments, the viscosity of the polymer solution ranges from about0.10 centipoise to about 100,000 centipoise, or any viscosity inbetween, including 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300,400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000,8000, 9000, 10000, 20000, 30000, 40000, 50000, 60000, 70000, 80000,90000, or 100,000 centipoise. In other embodiments, the viscosity of thepolymer solution ranges from about 1 to about 1,000 centipoise, or fromabout 1 to 7 Pa*s, such as from about 1 to 3 Pa*s. In other embodiments,the viscosity of the polymer solution ranges from about 1 to about 100centipoise. By way of illustrative examples, a solution of about 1centipoise has the viscosity of water, a solution in the hundreds ofcentipoise has the viscosity of motor oil, a solution of about 1000centipoise has the viscosity of glycerin, and a solution of about 50,000centipoise has the viscosity of ketchup. However, it is preferred thatthe viscosity of the polymer solution is maintained low enough so thatit is not too viscous such that the injection cannot be performed with asyringe and needle. The viscosity of the polymer solution can bemanipulated by the varying the polymer and/or solvent chosen, thepolymer concentration, polymer molecular weight, microbubblecomposition, microbubble size, microbubble concentration, andcross-linking.

In embodiments, the substance, solution, or composition administeredinto the body lumen can be a composition comprising a polymer,copolymer, block copolymer, monomers, or a polymer precursor orpre-polymer, wherein the composition is capable of forming a polymerocclusion in the body lumen upon administration therein. In some cases,polymerization of the substance, solution, or composition may occurbefore, during, and/or after administration of the substance, solution,or composition into the body lumen, such as a vas deferens. Further, forexample, in the context of this specification referring to administeringa polymer can include administering a polymer already formed, oradministering the precursor or pre-polymer to a copolymer or blockcopolymer, such as polymer(s) or copolymer(s), or administering theprecursor or pre-polymer to a polymer, such as one or more monomers, orcombinations thereof, whether or not the monomer(s), polymer(s), orcopolymer(s) have already begun forming the polymer, copolymer or blockcopolymer prior to, during, or after administration of the composition,solution, or substance. In situ forming of the occlusion in a bodylumen, such as a vas deferens, includes methods where any part of theforming of the occlusion occurs in the body lumen and not necessarilythat the occlusion must be formed completely in situ. Thus, forming theocclusion completely or partially in a body lumen are included asaspects of embodiments of the invention.

In embodiments, other constituents are included in the vas-occlusivepolymer solution. These may include additional contrast agents, imagingagents, therapeutic drugs, antimicrobials, anti-inflammatories,spermicidal agents, vasodilators, steroids, hormones, ionic solutions,proteins, nucleic acids, antibodies, or fragments thereof. The otherconstituents can provide additional contraceptive activity to thevas-occlusive polymer solution. For example, the other constituents mayproduce an effect on sperm motility, viability, or fertility and may bea small molecule, protein, peptide, antibody, nucleic acid, or fragmentthereof. For example, a 22 kD sperm protein, SP-22, correlates withfertility and predicts fertility in males. See U.S. Pat. No. 6,197,940.Antibodies to this protein, such as those described in U.S. Pat. No.7,754,212 can be included in the vas-occlusive polymer solution.Antibodies against other sperm proteins can be included. In one aspect,the antibody is S19, also known as MHS-8, against the sperm-specificglycoprotein SAGA1. Additionally, spermicidal agents such asnonoxynol-9, oxtoxynol-9, benzalkonium chloride, or chlorhexidine can beincluded. Additionally, the vas-occlusive polymer and/or solvent can beinnately spermicidal, such that no exogenous spermicidal agent isincluded.

In one embodiment, the vas-occlusive polymer may be modified orcross-linked with fusion proteins, amino acid sequences, or peptides(natural or synthetic). In one aspect, the polymer may be modified withpolyethylene glycol (PEG), where PEGylation may enhance thebiocompatibility of the polymer. The polymer may be modified with anamino acid sequence. In another aspect, the amino acid sequence containslysine residues which are cross-linked to the maleic acid or maleicanhydride groups. The amino acid sequence may be cleaved with an endo-or exo-protease. In one aspect, the amino acid sequence is a dipeptide.The addition of a protease causes the gel to de-precipitate, liquefy, ordissolve for reversal. In one aspect, the protease is found naturally inthe human body. In one aspect, the protease is not found in the humanbody. The amino acid sequence and protease may be chosen from adatabase. In one aspect, the protease is papain, bromelain, actinidin,ficin, or zingibain. In another aspect, the di-amino acid scission sitemay only be cleaved by a bacterial protease. Preferably, the protease isinjected in a solution form into the vas deferens to reverse,de-precipitate, liquefy, dissolve, or flush out the polymer gel.

In embodiments, the vas-occlusive polymer solution is formulated toinclude an ultrasound contrast-enhancing agent such that is or becomesechogenic. The ultrasound contrast agent can be microbubbles, or anyother known ultrasound contrast agent or which becomes known in the art.Ultrasound contrast agents have been reviewed in the literature (seeCalliada F, et al., “Ultrasound contrast agents: basic principles”, EurJ Radiol. 1998 27 Suppl 2:S157-60 and Cosgrove D, “Ultrasound contrastagents: An overview”, Radiology 2006 Volume 60, Issue 3, Pages 324-330).In one embodiment, the microbubbles decrease the lateral and axialresolution (as calculated by the full-width, half-maximum formula),thereby enhancing the visibility of the gel on the ultrasound. Theaddition of microbubbles allow for the gel to be echogenic (visible onultrasound) for extended duration.

In embodiments, the ultrasound contrast-enhancing agent includesgas-containing microbubbles or microspheres. The microbubbles may alsobe hollow or porous. The gas may include a mixture or combination ofgases (e.g. air), or any inert gas, such as nitrogen, argon,perfluorocarbon, and the like. The microbubbles may have a shell whichincludes as components a polymer, a lipid, a protein, a surfactant, amonosaccharide, a polysaccharide, or glass. Provisional PatentApplication No. 62/254,381 disclosed a vas-occlusive polymer gelcontaining microbubbles (e.g., glass). The microbubbles caused the gelto be echogenic, or visible under ultrasound.

Useful polymers for microbubbles may include, but are not limited to,polystyrene, neoprene, polyetherether 10 ketone (PEEK), polyethylene,polypropylene, polyetherketoneetherketoneketone (PEKEKK), nylon, TEFLON®TFE, polyethylene terephthalate (PETE), TEFLON® FEP, TEFLON® PFA, andpolymethylpentene (PMP). The polymers may be insoluble in DMSO. Usefulpolysaccharides for microbubbles include, but are not limited to,cellulose, cellophane, or carboxymethyl cellulose, or any derivativethereof. An example of a protein constituent of a microbubble shellincludes albumin, and an example of a saccharide constituent of amicrobubble shell includes galactose. Additionally, the microbubbles mayinclude multiple constituents.

In embodiments, the microbubbles can be formulated to contain additionalagents or drugs, including, but not limited to, therapeutic drugs,antimicrobials, anti-inflammatories, steroids, drugs, hormones, ionicsolutions, proteins, peptides, antibodies, or nucleic acids, orfragments thereof. The additional drugs or agents can be controlledthrough sustained release. The molecules can be conjugated to themicrobubbles of the invention or included as internal components of themicrobubbles. The molecule may be a small molecule, protein, a peptide,antibody, or a ligand which targets sperm to render the sperm immotile,infertile, or inviable.

In embodiments, the microbubbles or microspheres of the invention canvary in size, or can be provided in a fairly uniform size range. Themicrobubbles can range in size from about 0.10 to about 1,000 μm indiameter. However, a substantially uniform size range is preferred toprovide maximum contrast. For example, the microbubbles can be providedin a size ranging from about 1-2, 2-3, 3-4, 4-5, 5-6, 6-8, or 8-10 μm indiameter. Additionally, the microbubble shell thickness can vary insize. In embodiments, the microbubbles are provided at a concentrationranging from about 1×10² to about 1×10⁹ microbubbles/ml, including1×10³, 1×10⁴, 1×10⁵, 1×10⁶, 1×10⁷, and 1×10⁸ microbubbles/ml. In someembodiments, the microbubbles are innately present in the vas-occlusivepolymer solution. In other embodiments, the microbubbles are fabricatedseparately and added to the vas-occlusive polymer solution.

For example, microbubbles or microspheres of the invention can becrosslinked to the polymer gel. The microbubbles or microspheres can beadded separately to the gel solution and mixed to form a homogenoussolution. The solution can be mixed by stirring. For example, themicrobubbles can be formed by mixing a polymer-DMSO solution to form afoam solution. In other embodiments, a double emulsion method can beused (see El-Sherif, D. M., & Wheatley, M. A. “Development of a novelmethod for synthesis of a polymeric ultrasound contrast agent”, Journalof Biomedical Materials Research Part A, (2003) 66A(2), 347-355), aswell as by pumping the polymer-DMSO mixture through 2 syringes and 3-waystopcock, a method which has been employed for producing agitatedmicrobubbles, but only in saline solutions (see Attaran, R. R, “Protocolfor Optimal Detection and Exclusion of a Patent Foramen Ovale UsingTransthoracic Echocardiography with Agitated Saline Microbubbles”(2006), Echocardiography (Mount Kisco, N.Y.), 23(7), 616-22). Using thedouble emulsion method according to the present invention, air-filledpolystyrene and polyvinyl alcohol (PVA) microbubbles were produced. Themixing can also be accomplished by transferring the solution betweensyringes (from one to another). Further, specific size ranges ofmicrobubbles can be isolated by differential centrifugation and added tosamples of vas-occlusive polymer solutions, and the samples may besubject to ultrasound imaging. In this way, the maximum echogenicity(contrast) of the microbubbles in different size ranges can bedetermined.

In one embodiment, microbubbles are prepared by pumping the polymer-DMSOsolution through 2 syringes connected by a 3-way stopcock with swivelmale luer lock. The number of pumps can be from 1-200, such as from 1-2,2-3, 3-4, 4-5, 5-6, and so on. The lateral and axial resolution of thegel decreases with number of pumps, indicating that more microbubblesare formed with increased number of pumps. The volume of air in thesyringe loaded prior to the pumps can be varied from 0-1 mL. In oneexample, performing 80 pumps with a loaded air-volume of 0.75 mL yieldsthe lowest lateral and axial resolution (highest visibility of the gel).This combination also yields the smallest decrease in visibility overtime. In some embodiments, the microbubbles that are formed have noshell and comprise air. Larger microbubbles are found at the top of thepolymer solution in the syringe. In another aspect, the smallermicrobubbles are found at the bottom of the polymer solution in thesyringe. In embodiments, the polymer-DMSO-microbubble solution isinjected into a bodily duct (e.g., vas deferens).

In one embodiment, the microbubbles are prepared through the followingdouble emulsion procedure. The polymer of interest is dissolved inorganic solvent such as chloroform. Water is added and the solution issonicated. Further, a surfactant such as PEG stearate is added toseparate the water droplets within the bulk of organic solvent andpolymer. The emulsion is poured into a polyvinyl alcohol (PVA) solutionand homogenized. Preferably, the PVA solution is 5%, but can range fromabout 1% to about 80%, such as from 1% to 2%, 2% to 3%, 3% to 4%, 4% to5%, 5% to 6%, 6% to 7%, and so on. The homogenized solution is pouredinto isopropanol. Preferably, the isopropanol solution is 2%, but canrange from around 1% to about 50%, such as from 1% to 2%, 2% to 3%, 3%to 4%, 4% to 5%, 5% to 6%, 6% to 7%, and so on. The mixture is stirredfor one hour at room temperature with stirring times that may vary. Themixture is then centrifuged to obtain a pellet of microbubbles. Thepellet is resuspended in water and re-centrifuged. The solution is thenlyophilized to remove the water from inside the microbubbles.

In one embodiment, perfluorocarbon gas (including perfluoromethane,perfluoroethane, perfluoropropane, perfluorobutane, perfluoropentane,and/or other such perhalocarbon gases) is contained within themicrobubbles, preferably as a contrast agent. In one aspect, doubleemulsion is used to prepare microbubbles containing perfluorocarbon gas.In one aspect, the gas is sonicated within a polymer and organic solventsolution. In one example, the emulsion is poured into around 5% PVA, butcan range from around 1% to around 50%, such as from 1% to 2%, 2% to 3%,and so on. In one aspect, the emulsion is poured into 2% isopropanol,but can range from around 1% isopropanol to around 35%, such as from 1%to 2%, 2% to 3%, and so on. In one aspect, the solution is allowed tostir at room temperature for around one hour. After around one hour, themixture is centrifuged, re-suspended in water, and centrifuged again toobtain a pellet of microbubbles. See U.S. Pat. No. 5,695,740.

In one embodiment, the microbubbles include polyvinyl alcohol (PVA). ThePVA bubbles can be cross-linked. The method by which the bubbles areprepared can include dissolving PVA in a solvent. In one example, anoxidizing agent is added, followed by double emulsion. The ends of thePVA polymeric chains are functionalized with aldehyde groups beforecontinuing double emulsion. See Chinese Patent No. 103724638 A and U.S.Patent Application Publication No. US 20100158813 A1.

In one embodiment, the shells of the microbubbles are made ofpolystyrene. In one aspect, the molecular weight of the polystyreneranges from 35,000-400,000 daltons, such as from 35,000 to 40,000daltons, from 40,000 to 45,000 daltons, from 45,000 to 50,000 daltons,from 50,000 to 55,000 daltons, and so on.

In one embodiment, the microbubbles include one or more spermicides,such as those discussed herein. In one aspect, the microbubbles containa degradation agent such as a reducing agent (e.g., glutathione) toassist in reversal of the polymer gel.

According to embodiments, the vas-occlusive polymer solution isformulated to have a specific porosity once it polymerizes in situ toform a gel. For example, the porosity can be tailored to allow passageof fluids (as well as constituents such as proteins, nutrients, etc.) inthe vas deferens while blocking sperm cells. In embodiments, the porediameter is less than about 3 μm (e.g. the approximate width of the headof a human sperm cell). In embodiments, the pore diameter of the formedpolymer can range from 0.001 nm to 3 μm, such as from 0.001 nm to 1 μm.In other embodiments, the pore diameter can range from 0.01 nm to 100nm. In other embodiments, the pore diameter can range from about 1 nm toabout 1 μm. In other embodiments, the pore diameter can be 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.10, 0.15, 0.20, 0.25, 0.30,0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90,0.95, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,60, 65, 70, 75, 80, 95, 90, 95, or 100 nm. In other embodiments, thepore diameter is at least the size of an atom (0.5 nm). Specific poresizes can be targeted to provide an optimum porosity that providesmaximum flow of fluid while blocking the flow of sperm cells.

In embodiments, the vas-occlusive polymer solution of the invention isadministered into the vas deferens at a rate or amount which dictatesthe shape and length of the vas-occlusive plug that forms. The rate ofadministration can be constant or variable. For example, thevas-occlusive polymer solution can be injected or infused at a rate of0.001 cc/min (1 μl/min) to 1.0 cc/min (1 ml/min), including 0.001,0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02,0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.15, 0.2, 0.25, 0.30,0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.90,0.95, and 1.0 cc/min. It is preferable that the physician injects thepolymer material at a constant rate. With a relatively slow injectionrate (e.g. about 0.1 to 0.2 cc/min), the polymer will form atightly-packed, cylindrical gel. At a relatively fast injection speed(e.g. greater than about 0.50 cc/min), the polymer gel can be morestring-like and may not fully occlude the vas. A pressure-controlledsyringe or device may be used to ensure a constant injection speed.

Total volumes administered can vary from about 1 μl to about 1000 μl (1ml), including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40,45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140,150, 160, 170, 180, 190, 200, 225, 250, 275, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950, and 1000 μl.

The rate of administration and total volume administered can depend onthe size of the vas deferens in terms of diameter and length of thelumen, as well as the composition and properties of the polymer solutionin terms of molecular weight and concentration of the polymer,viscosity, gelation temperature, rate of polymerization, and desiredlength of the occlusion. Such is within the capabilities of a skilledartisan. In one aspect, the volume and rate injection is low enough thatthe polymer does not leak into the vas wall or rupture the vas.

In embodiments, the length of occlusion produced in the vas deferens asa result of administering the occlusive substance can range from 0.1-5centimeters in length, including 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2,2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, and 5.0cm in length. In embodiments, the length of the occlusion formed in thevas deferens is longer than the smooth muscle fibers of the vas deferensso that the plug is not dislodged by peristaltic contraction.Preferably, the polymer gel plug formed in the vas deferens is rigidenough so that the peristaltic contraction of the vas deferens does notbreak, dislodge, or otherwise affect the safety and/or efficacy of thepolymer gel. According to one embodiment, the injection volume andlength of polymer plug that forms is determined such that the subjectbecomes severely oligospermic or azoospermic as determined by analysisof semen samples. Upon administration, polymerization of thevas-occlusive polymer can be monitored in real time using ultrasound.

In embodiments, ultrasound is used to image the vas-deferens and thevas-occlusive polymer during and after placement inside the vasdeferens. Ultrasound based imaging is a painless and convenientdiagnostic method that functions by projecting sound waves into thebody, and then measuring the refraction, reflection, and absorptionproperties of the imaged-tissue to assess fine structure. Essentially,the way in which certain structures reflect sound waves allows for thegeneration of an image of the underlying organs and tissues. Forinstance, ultrasound imaging works best on mechanically more elastic,sound conducting tissues. Calcifications in the body (such as bone,plaques, and hardened tissues) provide degrees of acoustic impedancethat makes it difficult to image structures lying below them.

Ultrasound is an ideal candidate for imaging the tissues in the malereproductive system. First, ultrasound imaging is non-invasive and safe.There is no associated ionizing radiation produced with ultrasound asfound in X-Ray, PET, and X-Ray imaging. Second, the male reproductivesystem, specifically the scrotum, does not contain bone, plaques, orhardened tissues which limit acoustic impedance. Finally, preparing apatient for ultrasound imaging is as simple as shaving the area ofinterest, cleaning the area of interest, applying anultrasound-conducting fluid interface gel to the surface of the skin,and applying the ultrasound probe in the correct orientation andposition. Therefore, ultrasounds are commonly found in urology clinicsand are used primarily for imaging the scrotum and penis.

Various frequencies can be used for imaging the vas deferens and/or gel,including contrast-pulse sequencing mode (7 MHZ), B-Mode imaging (14MHZ), and frequencies in between. Other possible ultrasound modes thatcan be used for the inventive methods include 2D mode, fusion, harmonicimaging (THI), color mode or color power angio, CW doppler mode, PWdoppler mode, M-Mode, anatomical M-mode (live or frozen image), B-Mode,color tissue doppler, PW tissue doppler, panoramic imaging, 3D/4Dimaging, and dual imaging. In some embodiments, the frequencies arebetween 1 and 20 MHZ, including 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, or 20 MHZ. Additionally, the ultrasound canbe delivered at different intensities, such as between 0.1 to 1 W/cm²,including 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and 1.0 W/cm².Additionally, the ultrasonic energy can be delivered at a specificpower, such as 0 to 20 Watts of energy, including 0, 0.1, 0.2, 0.3, 0.4,0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20 Watts. Additionally, the ultrasonic energy can bedelivered in pulsed or continuous mode. The ultrasound can be deliveredthrough an ultrasound unit. The ultrasound unit can be portable. Anexample of a portable ultrasound unit for scrotal imaging is the LOGIQV2, manufactured by GE Healthcare (Little Chalfont, United Kingdom).Another example of an ultrasound unit for scrotal imaging is theClearVue 350 by Philips (Amsterdam, Netherlands).

According to embodiments, various ultrasound probes or transducers canbe used for ultrasound imaging the vas deferens, including sector(phased array), linear and convex transducers. Ultrasound probes andtheir selection have been discussed in the literature (see T. L. Szaboet al., “Ultrasound Transducer Selection in Clinical Imaging Practice”,Journal of Ultrasound in Medicine, 2013, 32(4):573-582). Ultrasoundtransducers differ according to their piezoelectric crystal arrangement,physical dimensions, shape, footprint (aperture), and operatingfrequency. It is within the ability of a skilled artisan (e.g. urologistor ultrasound technician) to choose a transducer with appropriatecharacteristics to image the area of the vas deferens that has beenisolated. A hand-held probe may be chosen for imaging that is smallenough to image the vas without interfering with other aspects of theprocedure such as administration of the occlusive substance.

Transducers are multi-frequency, meaning the frequency can be switchedelectronically between a range of frequencies (e.g. abdominaltransducers have 2-6 MHz). It is important for the user to select thehighest frequency which adequate depth of penetration for the anatomicarea of interest. In general, the higher the frequency of thetransducer, the greater than axial resolution and better the anatomicrepresentation of the image. However, there is a tradeoff betweenfrequency and depth of penetration. For imaging the testis, because ofthe close proximity of the organ to the surface of the skin, imaging canbe performed with high frequency transducers such as a linear arraytransducer of 12-18 MHz.

There are many factors that impact the image quality. Parameters andsettings may be modified by the user of the ultrasound in order toadjust and manipulate the image including: gain, time-gain compensation,frequency, depth/size, field of view, and cine function. A “good qualityimage” includes: (1) sufficient and uniform brightness, (2) is sharp andin focus, (3) adequate size, and (4) is oriented and labeled fordocumentation purposes. Furthermore, selection of a transducer iscritical for maximizing image quality. Linear array transducer probesproduce a rectangular image whereas a curved array transducer produces atrapezoidal shape. Linear array transducers are most commonly used inurology for imaging the testes and male genitalia. However, a curvedarray transducer can be helpful in visualizing both testessimultaneously.

In regards to safety, the FDA advises that the mechanical index (MI) andthermal index (TI) are kept below 1.90 and 6 degrees C., respectively.

This disclosure reports that ultrasound is the ideal imaging modalityfor performing a guided injection into the vas deferens. The relativelyshallow depth at which the vas deferens sits allows for easyidentification by a medium or high frequency ultrasound. Ultrasound israrely used in clinical applications for imaging the vas deferens. Thus,prior to the present disclosure, methods for optimal imaging of the vasdeferens were limited. To the best of the knowledge of the presentinventors, ultrasound-guided, percutaneous injection into the vasdeferens has never been performed. Optionally using ultrasound asguidance for performing percutaneous vas injections is critically neededbecause: 1) every subject has different morphometric measurements of thevas (e.g. outer and inner diameter, depth, length), 2) the physician orother professional (e.g. technician, veterinarian, etc.) performing theprocedure can visualize that the needle is inside the vas lumen asopposed to the smooth muscle layers of the vas, 3) the physician canvisualize the polymer solution being injected into the lumen, 4) thephysician can visualize the polymer forming a hydrogel in situ in realtime, 5) the physician can observe the length of the gel plug,confirming that enough of the material was injected, 6) the physiciancan perform routine “check-ups” on the composition using ultrasounddays, weeks, or months after the composition is inserted, 7) thephysician can locate the gel prior to reversal, 8) the physician canperform the reversal through ultrasound-guided, percutaneous injection,and 9) the physician knows that the reversal was successful if the gelis no longer visible on the ultrasound.

According to embodiments, the physician isolates the vas deferens usinga finger technique and secures it to the scrotal skin using a vas clamp.In one embodiment, an ultrasound probe is placed on the vas deferensbefore, during, and/or after administration of the occlusive substance.The probe can be placed parallel to the vas such that the lumen will bevisualized with a longitudinal view. In this view, the length of the vascan be determined as well as the inner and outer diameter. Alternativelyor in addition, the probe can be placed perpendicular to the vasdeferens such that the lumen of the vas will be visualized inaxial/transverse view. In this mode, it is easy for the physician todiscern the depth of the vas as well as determine the inner and outerdiameter length.

According to one embodiment, the physician administers a vasodilatorlocally in the area of the secured vas deferens prior to administrationof the occlusive substance. The vasodilator diffuses to the smoothmuscle of the vas and causes it to relax, thereby expanding the lumen toa dilated state. This pharmacologic dilation of the vas can facilitatethe procedure by 1) providing greater visibility of the lumen of the vasunder ultrasound and 2) providing a larger target for insertion of aneedle or catheter into the lumen, thereby reducing the chances of“off-target” insertion into the smooth muscle of the vas. Vasodilatorsare known in the art, including nitric oxide donors (e.g.nitroglycerin), acetylcholine, prostaglandins, papaverine, calciumchannel blockers, phosphodiesterase type 5 (PDE-5) inhibitors, and thelike. The vasodilator can be administered with an anesthetic agent or bepresent in the solution of the vas-occlusive substance. In oneembodiment, the vasodilator is administered percutaneously. In otherembodiments, the vasodilator is administered topically. In otherembodiments, no vasodilator is administered.

According to embodiments, a local anesthetic agent is administered inthe area of the vas deferens prior to administration of the vasocclusive substance. Various local anesthetic agents are known,including amino esters such as procaine (Novocaine), tetracaine(Pontocaine), benzocaine, as well as amino amides such as lidocaine,mepivacaine, bupivacaine, and etidocaine. To avoid constriction of thevas deferens lumen, it is preferred that the local anesthetic agent besubstantially devoid of vasoconstrictive activity, and that novasoconstrictive agents such as epinephrine be administered with theanesthetic. The anesthetic can be administered topically orpercutaneously.

Once the lumen is visualized, the physician percutaneously inserts aneedle, cannula, catheter, or needle-catheter into the lumen underoptional guidance of ultrasound imaging and administers (e.g. injects)the occlusive substance (e.g. polymer solution) into the lumen. Theinjection process may be performed towards the direction of the testes,which would be against the flow of seminal fluid and assist in gelformation, or in the direction of the prostate. The region of the vaswhere the injection can be performed is either in the scrotal region orsupra-scrotal region before the vas extends into the pre-pubic region.An echogenic needle can be used, which facilitates visual confirmationthat the needle is inside the lumen via ultrasound beforeadministration. Alternatively, an ultrasound with magnetic field needleguidance such as the eZono® 4000 (eZono AG, Jena, Germany) can also beused to guide the needle more precisely into the lumen in axial mode.The ultrasound may also be a handheld, portable ultrasound.

If the polymer is echogenic, it will be seen being injected into thelumen. Then, the physician will be able to witness the polymer form ahydrogel in the vas deferens in real-time.

After the gel “plug”, or occlusion, forms, the physician can confirm theprocedure was done properly by ultrasound imaging the gel. Axial modecan be used for viewing to determine if the gel completely occluded thevas, whereas longitudinal mode can be used determining the length of thevas-occlusive gel plug. In this way, the dimensions (e.g. length, width,and diameter) of the occlusion can be determined through ultrasoundimaging. If the occlusion is not of sufficient size in terms of diameterand length, additional material can be administered inside the vasdeferens lumen, until the physician confirms administration of anappropriate amount of vas-occlusive material through ultrasound imaging.

Thus, one particular embodiment of the invention provides a method ofvas-occlusive contraception which includes:

Non-surgically or surgically isolating the vas deferens in the scrotumsuch as by using a three-finger type technique;

Optionally, administering anesthesia to the subject, such as byadministering local anesthesia to the vasal nerve region (e.g. vasalblock);

Raising the vas deferens and securing it, such as by securing the vasdeferens to the scrotal skin using a vas-clamp, making the vas assuperficial as possible;

Placing an ultrasound probe on the vas deferens and administeringultrasonic energy to the vas deferens to visualize the vas deferenslumen in longitudinal and/or axial view; and, by way of ultrasoundimaging:

-   -   Measuring one or more dimensions of the vas deferens lumen;    -   Placing a needle into the vas deferens and confirming placement        of the needle or catheter or a portion thereof into the lumen;    -   Percutaneously administering a vas-occlusive polymer solution        into the lumen;    -   Confirming formation of a polymer occlusion inside the lumen;        and    -   Determining one or more dimensions of the occlusion in        longitudinal and/or axial view.

The above procedure can then be repeated on the contralateral side.

For example, FIG. 1A is an illustrative diagram showing an embodiment ofa procedure of the invention in which a vas clamp is used to hold thevas deferens close to the scrotal skin. First, the general area of thescrotum is shaved and an ultrasound-conducting fluid interface gel isapplied to the scrotum (not shown). Then, an ultrasound probe is placedover the vas, ultrasonic energy is administered, and the physicianpercutaneously injects the vas-occlusive substance into the vas deferensoptionally under the guidance of ultrasound imaging. FIG. 1B is anillustrative diagram showing occlusion of the left vas deferens by wayof a vas occlusive plug after the procedure is performed such that spermcells are blocked from progressing through the lumen of the vasdeferens.

It is predicted that the inventive methods will be significantly quickerthan a typical vasectomy due to the fact that no incision orexteriorization of the vas is required. Furthermore, the inventivemethods skip the step of removing the sheath that surrounds the vas,which occurs during vasectomy (also adding a layer of safety which is ofissue with surgery).

As discussed herein, different aspects of the procedure include lengthof the procedure, the use of local anesthesia, using a vas-clamp orholding vas superficially to skin with fingers, rate of injection orinfusion, needle gauge size and/or length, syringes that can be used,vas deferens characteristics, such as depth of vas from skin, left orright vas, inner and outer diameter of vas, length of vas that isvisible, which portion of vas is administration performed, whichdirection is the administration performed (towards testes or prostate),and ultrasound properties, such as machine specs, probe specs,frequency, intensity, depth, mechanical index, and gain. Such are withinthe capabilities of a skilled artisan.

The amount of occlusive substance (such as an echogenic gel) to beadministered to a subject can vary based on several different criteria,including the duct or vessel where it is administered, the size of thelumen, the concentrations of the various components of the substance asadministered, the molecular weight of the polymer in the gel, the volumeof the gel solution that is administered, and the size of the occlusionthat the physician or person who is administering the gel solution istrying to achieve.

For example, ultrasound imaging of the vas deferens is particularlyimportant for determining the dimensions of its lumen, which determinesthe amount of substance administered. Administration of an insufficientamount of substance can result in the vas deferens only being partiallyoccluded, while administration of too much substance (particularly at ahigh rate) can rupture the vas deferens. Thus, according to oneembodiment, ultrasound imaging is used to determine the morphometricsand dimensions of the subject's vas deferens, including the innerluminal and outer total diameters, thickness of tunics, and length ofthe vas deferens. As use of the method increases, a robust database canbe generated that will provide valuable information regarding theanatomical and physiological feature of the human male reproductivetract. This new information could potentially further novel advancementsin male reproductive health, and will help to standardize knownanatomical information on the vas deferens. In 107 men in China, it wasreported that the average inner diameter was 0.56 mm and the averageouter diameter was 2.17 mm (Liu, X. et al.). However, various otherstudies have shown that the inner diameter can be as little as 0.31 mmand can dilate as large as 1.8 mm (Liu, X. et al.). The injection volumeof the occlusive agent that should be delivered to each individual is afunction based on several criteria including: the size of theindividual's vas lumen, the concentration of the polymer, the molecularweight of the polymer, monomer ratio of the polymer, injection speed,and desired plug length. Ultrasound allows for the determination of thepatient's lumen and plug length. The same paper by Liu et al., reportsthat the mean rupture volume is around 0.05 mL for 1 cm of vas. Duringthe inventive procedure, ultrasound can be used to ensure that thesubstance has occluded the lumen and that additional material should notbe injected in order to prevent rupturing of the vas.

The vas-occlusive substance (e.g. polymer solution) can be administeredinto the as lumen by hand through a standard hypodermic needle andsyringe, such as those manufactured by Becton Dickinson (Franklin Lakes,N.J.). In one embodiment, an injection device is used for the procedurewherein said device maintains an almost constant injection speed andvolume during the injection. In one embodiment, this injection device isa pressure-controlled syringe. The polymeric composition can be providedin a pre-filled syringe, vial, or other suitable container.Alternatively, the vas deferens can be cannulated or catheterized by wayof insertion of tubing and a vas-occlusive polymer can be administeredmechanically through the use of an infusion pump, such as thosemanufactured by Cole-Parmer (Vernon Hills, Ill.). The use of an infusionpump facilitates precise, controlled flow rates and quantities ofvas-occlusive polymer solution into the vas deferens lumen.

In embodiments, the vas-occlusive polymer is monitored at various timesfollowing administration. For example, it can be monitored usingultrasound at various times, including, days, weeks, months, and yearsafter administration to determine whether it is still there and tomonitor its integrity. Monitoring is useful for determining that thevas-occlusive polymer has polymerized to form a gel, the location of thegel, stability of the gel, effectiveness of the gel, and longevity ofthe gel, as well as its use as a contraceptive. In addition, ejaculatesof the subject can be monitored and sperm counted and measured forviability, motility, activity, etc. and compared to the ultrasoundresults. Such monitoring can determine the need for a follow-upprocedure, such as re-administration of the vas-occlusive polymer to thesubject.

In embodiments, the longevity or stability of the polymer gel isestimated by counting the number or concentration of microbubbles insidethe gel. The longevity or stability of the polymer gel can be furtherestimated by determining the echogenicity of the polymer gel usingultrasound. Alternatively, the longevity or stability of the polymer gelcan be evaluated by observing the shape, size, or attachment of thepolymer gel to the lumen of the vas deferens by way of ultrasound.

In embodiments, solutions of the invention are formulated withmicrobubbles which incorporate an agent which renders sperm immotile,infertile, or inviable. The agent can be incorporated inside themicrobubbles or conjugated to the microbubbles.

Another embodiment of the invention is a method of delivering of anagent to the lumen of the vas deferens. The method includesnon-surgically or surgically isolating the vas deferens of a subject,administering a solution into the lumen of the vas deferens, andapplying ultrasonic energy at a frequency which is capable of lysingmicrobubbles present in the solution, thereby releasing the agent intothe lumen of the vas deferens. Alternatively, the microbubbles may beallowed to slowly dissolve without the use of ultrasound such that theagent is released to the lumen at a constant rate over time. In thisway, the vas-occlusive polymer provides both a physical barrier to thepassage of a sperm as well as targeted inhibition of sperm cells.

For example, in one embodiment, focused ultrasound is applied at aparticular frequency which causes the microbubbles to vibrate. At aparticular threshold of intensity and/or frequency, the microbubbles canbe destroyed, which can cause a local shock wave, resulting incavitation and lysing of the gel. Thus, the use of ultrasound canprovide a non-invasive method of reversing the vas-occlusivecontraception provided by the invention. Accordingly, one embodiment ofthe invention provides a method of reversal of a vas-occlusivecontraception comprising applying ultrasonic energy to a vas-occlusivegel plug at a frequency and/or intensity that is capable of destroyingmicrobubbles inside the vas-occlusive gel plug, thereby lysing anddestroying the occlusion.

In one embodiment, a level of ultrasonic energy needed for microbubblecavitation is determined. For example, a detector transducer receives ascattered level of ultrasonic energy, indicative of stable cavitation.Accordingly, a method for in vitro or ex vivo testing of microbubblecavitation is used to determine acoustic pressures necessary forreversal. In one aspect, the gel with microbubbles is precipitated indialysis tubing. By way of example, the gel with microbubbles isprecipitated in an excised vas deferens or synthetic vas deferenstissue, and an ultrasound probe is applied at varying frequencies,wherein for each frequency, the amount of gel lost is measured. Once ameasurement is recorded which is expected to adequately reverse,de-precipitate, liquefy, dissolve, or flush out the polymer gel, such afrequency can be used to reverse, de-precipitate, liquefy, dissolve, orflush out the polymer occlusion in a subject.

An additional embodiment of the invention includes a method of reversalof a vas-occlusive contraception comprising non-surgically or surgicallyisolating the vas deferens and administering a solvent or solution inthe lumen of the vas deferens that is capable of dissolving avas-occlusive polymer plug disposed in the lumen of the vas deferens.For example, the method of reversal may rely on ultrasonic imaging todetermine the location of the vas-occlusive polymer plug in the vasdeferens. Then, the vas deferens may be isolated according to thethree-finger technique and use of a vas-clamp as previously described.Then, a solvent or solution which is capable of dissolving the polymermay be administered into the lumen of the vas deferens by way ofpercutaneous injection. Alternatively, the solvent or solution can beused to “flush out” the occlusion. For example, the solvents can includeDMSO and the solutions can include sodium or potassium bicarbonate. Inone aspect, the solution has a pH from 8-9. As an alternative tobicarbonates, other alkaline solutions can be used. Anywhere from 0.01-3cc of solvent or solution can be injected into the lumen of the vasdeferens, such as 0.01 to 0.02 cc, 0.02 to 0.03 cc, 0.03 to 0.04 cc, andso on. However, the rate and volume of injections are limited such thatthe injection force does not rupture the walls of the vas deferens. Thedissolution of the polymer occlusion can then be monitored in real timeusing ultrasound. Absence of the occlusion and patency of the vas lumencan be confirmed via ultrasound imaging, and ejaculates can be monitoredpost-procedure to determine restoration of sperm counts in theejaculate. In this way, successful reversal of contraception can beconfirmed.

In some embodiments, reversal of contraception is performed surgically.The vas deferens can be exteriorized, a small slit can be made, and theocclusion may be able to be pulled out (especially in the case ofsilicone devices). In some embodiments, the occlusion may bemechanically reversed using a miniature drill. If these methods areineffective, the segment of the vas containing the gel may be ablatedand re-anastomosed in a procedure identical to vasovasostomy.

According to additional embodiments, compositions and methods areprovided that provide for radio-frequency, photoacoustic, and/orinfrared detection of the occlusive substance in addition to ultrasound.For radio-frequency detection, particles include but are not limited togold or radiofrequency identification (RFID) powder, both of which canbe recognized by a recognition device/reader. RFIDs come in the form ofchips or elements that are subjected to a radio frequency by a readerprobe. Different RFIDs have been manufactured, including RFID powder,which has a surface area of around 0.3 mm² (see U.S. Pat. Nos.8,440,487; 8,766,853; and 8,933,784). The powder may be added to avas-occlusive polymer solution and incorporated into the contraceptiveonce the gel is formed. Similarly, a dye could be incorporated into thecontraceptive for infrared imaging.

Thus, embodiments provide occlusive substances that are (1) effectivelong-term, (2) reversible, and (3) detectable by (i) ultrasound, (ii)radio-frequency, (iii) infrared, and/or (iv) photoacoustic imaging.Embodiments of the present invention include an occlusive substancewhich includes at least one polymer, a solvent medium, and at least oneimaging agent, such that the occlusion is detectable when the substanceis injected into a body cavity or lumen. Fields of use include, but arenot limited to treatment (e.g., occluding) of the vas deferens,fallopian tube, aneurysms, blood vessels, ducts, tumors, and organs.

In one embodiment, the polymer is modified by adding or cross-linking adye including but not limited to fluorescent dyes (e.g., atto680). Inone aspect, the polymer gel modified with the dye can be reversedthrough exposure to a laser.

In one embodiment, the occlusive polymer gel contains microbubbles,radio-frequency detectable particles, ultrasound-detectable particles,photoacoustic-detectable particles, or a combination of them.

In one embodiment, the polymer gel contains particles that allow for thegel to be detectable with radio-frequency. For example, the particlesmay be gold nanorods, or the particles may be RFID chips, elements,powder, or some other means of imparting RFID properties to the polymergel. In one aspect, the RFID is active or passive. In one aspect, if theRFID is active, it could be written on as well as read. In one aspect,if the RFID is active, it could be functioning constantly. In oneaspect, if the RFID is passive, it does not require a battery.

In one embodiment, the gold nanorods encapsulated in the polymer gel areultrasound visible. In one aspect, the gold nanorods have a polymercoating. In one aspect, the polymer coating is functionalized with anantibody or DNA. In one aspect, the gold nanorods have a silica coating.In one aspect, the gold nanorods can be excited with photoacousticenergy.

In a preferred embodiment, the RFID properties are imparted to thepolymer gel in the form of RFID powder. The RFID powder may bedistributed or added to the polymer solution such that when a gel forms,the RFID element would be dispersed within the gel composition. In oneaspect, the RFID powder is 0.05 mm×0.05 mm×0.0005 mm. In one aspect, theRFID powder responds to 2.45 GHz frequency. In one aspect, the RFIDpowder has a 128-bit ID. The RFID powder may be placed or distributed atdifferent points inside the polymer gel, wherein it may be dispersedrandomly, non-randomly, uniformly, non-uniformly, or in some othermanner. In one aspect, the RFID is fixed into place inside the gel. Inone aspect, tens, hundreds, thousands, or more RFID powder granules orelements are implanted and/or dispersed inside of the polymer gel. Inone aspect, different gauge needles are used to implant differentamounts or sizes of RFID elements. In a preferred embodiment, a 34 gaugeneedle is used to implant the RFID, and the RFID element or powder isplaced inside of the vas deferens.

In one aspect, the RFID element or RFID powder is contained withinanother substance or material, such as a casing or solution; forexample, to make the RFID more biocompatible. In one aspect, the casingcan be visualized, identified, or otherwise detected using ultrasound.In one aspect, the casing does not severely interfere with the tag'sresponse or reading. In one aspect, the casing is chemical resistant tothe gel that surrounds it. In one aspect, the casing material is inert.Preferably, the casing is not so thick that the RFID element is nolonger functional or that the gel no longer has RFID properties.

In one embodiment, the silicon substrate that is the base of the RFIDchip is replaced with a glass substrate, making the tag ultrasoundvisible.

In one embodiment, RFID technology is used to detect the presence of asubstance within a body, including a vas-occlusive gel or some otherocclusive gel. In one aspect, RFID technology is used to detect theformation of a polymer gel occlusion in the body. In one aspect, thepolymer gel occlusion to be detected by RFID technology is inside thevas deferens. In one aspect, the polymer gel occlusion is inside thefallopian tubes. In another aspect, RFID technology is used to detectthe presence, stability, longevity, effectiveness, and/or other propertyof an occlusive polymer gel hours, days, months, or years after thepolymer gel is implanted. RFID technology may be used to detect changesin the presence, stability, longevity, effectiveness, and/or otherproperty of a bodily substance over time.

In one embodiment, RFID technology is used to detect degradation of asubstance in the body. In one aspect, said substance is a polymer gel.In one aspect, the degradation can be tracked over time. In one aspect,each tag contains a rectifier circuit which converts the signal intopower for the device. In one aspect, the circuit can be used to powerother devices. In one aspect, the RFID element can be activated torelease a degrading agent, such as, for example, a reducing agent (e.g.,a peptidase). In one aspect, RFID technology can be used to trigger therelease of spermicide, antibacterial agents, or steroidal agents.

In one embodiment, RFID technology is used to detect whether the polymergel was reversed. For example, when the polymer gel is reversed,de-precipitated, liquefied, dissolved, or flushed out, the RFID elementsare excreted through urination or other means of removing foreign bodies(e.g., RFID elements) from the body. In one aspect, reversal isconfirmed by the lack of RFID tag response.

In one embodiment, a multi-tag system using RFID technology is used. Inone aspect, multiple RFID frequencies are used to differentiate tags. Inone aspect, different frequencies are used to differentiate segments ofa substance (e.g., polymer gel). In one aspect, multiple tags are usedas backups. In another aspect, a multitude of tags are used to generatea higher response.

In one embodiment, different subjects can receive polymer gels havingdifferent RFID tags in order to, for example, differentiate subjects. Inone aspect, this method is used to track patients. In one aspect, sinceeach RFID has 128-bits of information, 238 different IDs can be created.In one aspect, each subject can have their own RFID tag or otheridentifier.

In one embodiment, a reader is used that can detect RFID technology orelements through human tissue. In one aspect, the reader can detect RFIDpowder. In one aspect, the reader can detect gold nanorods. In oneaspect, the reader can detect RFID elements (e.g., powder) within ordispersed in a polymer gel. In one aspect, the reader can detect RFIDelements (e.g., powder) within a polymer gel within a bodily duct (e.g.,vas deferens). In one aspect, the RFID reader can examine, read, ordetect the polymer gel within the vas deferens with less interferencewhen the vas deferens is superficial to the scrotal skin.

In one embodiment, the reader can be an attachment for phones,computers, tablets, portable electronic devices, etc. In one aspect, thereader attachment would rely on the phone, computer, tablet, portableelectronic device, etc. to process and/or analyze the data, while thereader may, for example, only record raw data. In one example, thereader is personalized so that it can only work for a certain RFID tagor person. In one aspect, a reader is designed for detecting and/ortracking the presence, stability, effectiveness, longevity, and/or otherproperties of a polymer gel containing RFID technology (e.g., RFIDpowder). In one aspect, the reader attachment allows the user to trackhis/her RFID implant, without visiting a physician or other healthcareprofessional.

In one embodiment, the gel can be imaged by an infrared detectiondevice. In one aspect, the polymer gel contains an infrared dye, such asa clinical dye ICG. In one aspect, the polymer gel contains gold forinfrared detection. In one aspect, a laser is used at low power toexcite the fluorescence for detection. In one aspect, the excitation isat a 750-800 nm range. In one aspect, the emission filter is set with20-60 nm shift. In one aspect, the infrared detection device is acamera. In one aspect, the detection device has a laser pointer. In oneaspect, the detection device has a mated interference filter. In oneaspect, the detection device has a camera chip. In one aspect, thecamera chip is connected to a phone, computer, laptop, or otherelectronic device. In one aspect, the signal from the pigment of theskin is attenuated.

The methods and compositions of the invention can be used to providelong-lasting yet reversible contraception for human males, as well asmale animals such as pets, farm animals, zoo animals, and wildlife. Themethods have numerous advantages over other forms of contraception suchas vasectomy or neutering in terms of reversibility, costs, ease ofadministration, and lack of complications. Further, as the methodinvolves a one-time administration of a long-lasting contraceptiveagent, the method lacks the issues associated with contraceptive drugsor hormones such as adverse effects and lack of compliance.

The methods offer several medical benefits over vasectomy due to thefact that they are significantly less invasive and do not requiresurgical ablation of the vas deferens. First, a percutaneous injectionhas been shown to be less painful than incision and exteriorization ofthe vas as during vasectomy and has less chance for hematoma andinfection. Secondly, it is believed that implanting a polymer hydrogelmay reduce the chance for granuloma formation; if the pores of ahydrogel allow fluids and small molecules to travel through, this mayprevent sperm from extravasating and anti-sperm antibodies may decreaseor build up at a slower rate. Thirdly, by allowing fluids to travelthrough or around the hydrogel, then hydrostatic pressure in the vasdeferens will decrease. The buildup of hydrostatic pressure in the vasand epididymis after vasectomy is believed to be a major cause ofpost-vasectomy pain syndrome. Pain from post-vasectomy pain syndrome isthought to be also caused by sperm granulomas. Altogether, the methodspotentially reduce the chance for a patient to develop granulomas,hematomas, pain, or post-vasectomy pain syndrome.

Further, around 6% of men who receive a vasectomy later undergovasovasostomy (or vasectomy reversal). Vasovasostomy are difficultmicrosurgery procedures that requires general anesthesia, is expensive,and long (˜3 hours). Research has shown that patients who have avasectomy reversal >5-10 years after vasectomy decrease their chance forhaving offspring from 95% to 65% for reasons including the buildup ofanti-sperm antibodies. Embodiments of the present invention providemethods of reversal that are similar to the contraceptive methods exceptinstead of a polymer solution being injected into the vas lumen, adifferent solution is injected percutaneously into the lumen thatde-precipitates, dissolves, or liquefies the occlusive substance. Thepresent reversal methods are significantly shorter and easier to performthan vasovasostomy. After reversal is performed, the physician mayconfirm the procedure was successful based on if the gel is imaginableor not on the ultrasound.

The following Examples describe particular implementations of theinvention. They are intended to further illustrate the invention andshould not be used to limit the scope of the invention.

Example 1: Imaging of the Human Vas Deferens with Ultrasound

Background:

The non-surgical procedure disclosed herein to inject the echogenic gelis novel and is significantly different from the no-scalpel vasectomy(the current gold standard) as well as the procedure used to administerRISUG and VASALGEL. To distinguish this minimally invasive procedurefrom the vasectomy or injection of other vas-occlusive contraceptives,this procedure from hereon in, is known as VASINTOMY™. In the no-scalpelvasectomy, the three-finger technique is used to locate the vas at whichpoint a local anesthetic is injected into the scrotum and the vas. Asmall incision is made and the vas deferens is exteriorized. The sheathsurrounding the vas is removed, and vas is ablated. After the ablation,the vas is placed back inside the scrotum. The procedure is performed onboth sides. The injection of vas-occlusive contraceptives (RISUG,VASALGEL) is similar to the no-scalpel vasectomy, except for theinjection of a gel into the lumen of a vas instead of ablating the vas.

A limitation of this procedure is that there is no way to visualize thegel or the injection. The lumen of the vas deferens is on average0.4-0.6 mm in diameter. It is impossible to know if the urologistinjected the gel correctly into the lumen, or if it was injected intothe smooth muscle layers of the vas. Finally, there is no way to locatethe gel in the vas post-procedure or monitor its effectiveness,stability, or longevity. Primary market research has also revealed thatthe main factor why men choose not to get a vasectomy is the surgicalprocedure.

Thus, a novel aspect of the invention is the VASINTOMY™ orultrasound-guided, percutaneous injection of an echogenic contraceptivesubstance. This procedure uses ultrasound imaging to guide the injectioninto the lumen, at which point the release and precipitation orpolymerization (or otherwise formation of a solid or an occlusion) ofthe gel can be visualized due to its echogenicity. The VASINTOMY™requires no incisions, scalpels, or sutures. In this procedure, thethree-finger technique can also be used to locate the vas and then alocal anesthetic is injected. Then the vas is attached to the scrotumusing a vas-clamp, making it as superficial as possible. The urologistplaces a standard ultrasound probe against the vas to see the lumen, andthen the needle is guided through the skin into the lumen. The needlebecomes visible under ultrasound once inside the lumen. Next, theinjection is performed and the echogenic gel is released. Once formed orprecipitated, the gel can be imaged again to guarantee the procedure wasperformed successfully. This procedure allows for real-time imaging ofthe contraceptive during the injection.

For proof-of-concept of the VASINTOMY™, an IRB was filed and approved byDr. Ryan Smith, a urologist at the University of Virginia UrologyClinic. In this IRB, Dr. Smith clamped and imaged the vas of menreceiving the vasectomy procedure. If the lumen was visible underultrasound at high resolution, Dr. Smith was confident that he could dothe injection percutaneously.

Objectives:

1. Prove that the lumen of the vas deferens can be visualized with highresolution after clamping it to the scrotal skin

2. Observe the lumen in various views.

3. Determine the time for the urologist to observe the lumen with highresolution.

Methods:

The vas was isolated using the three-finger technique, local anesthesiawas injected into the area, and the vas was clamped to the scrotal skinor held the vas superficially to the skin using Dr. Smith's thumb. Then,Dr. Smith used a Philips HD XE 11 ultrasound with L15-7io probe (alsoknown as hockey-stick probe) and placed the probe longitudinally to thevas

Results:

In this clinical study, Dr. Smith from the UVA Urology Clinic tookultrasound images of the male patient's vas deferens prior to vasectomy.Dr. Smith stated that it took less than ten seconds to identify thelumen on the ultrasound. The ultrasound probe was positioned to view thevas in both transverse (FIG. 2) and axial view (FIG. 3). In both ofthese cases, the lumen could be seen clearly (marked by arrows).

The ultrasound image of FIG. 4 shows that the lumen of the vas deferensis distinguishable based on the fact that it has a black void runningthe length of the lumen compared to the tissue surrounding the vas.Using a measurement tool on the ultrasound (shown by plus signs), Dr.Smith measured the depth of the vas (from skin to mid-lumen) to bearound 0.4 cm or 4 mm. Thus, the vas is required to be as superficialfor high resolution, high-frequency imaging.

As shown in the ultrasound image of FIG. 5, Dr. Smith determined theinner lumen of the vas deferens to be 1.90 mm in the same patient. Thiscorresponds similarly to reported literature of the dilated inner lumendiameter.

As shown in the ultrasound image of FIG. 6, Dr. Smith was also able tovisualize the lumen of the vas deferens in the same patient using alower frequency probe L12-5. A length of 2.23 cm of the vas lumen couldbe visualized in this image. Thus, if a polymer gel was implanted duringthis imaging, up to 2.23 cm of the gel would be able to be visualized.

The image in FIG. 7 shows a Doppler ultrasound and color flow mapping ofthe artery that lies adjacent to the vas deferens in the spermatic cord.Color flow mapping using Doppler ultrasound allows real-time mapping ofblood flow patterns. Color Doppler ultrasonography allows for theevaluation of the velocity and direction of an object in motion. A colormap may be applied to the direction. The most common color map uses bluefor motion away from the transducer and red for motion towards thetransducer. The velocity of motion is designated by the intensity of thecolor. The greater the velocity of the motion, the brighter the colordisplayed. In urology clinics, Color Doppler is useful forcharacterizing blood flow in the kidneys, testis, penis, and prostate.In most clinical circumstances, the angle between the transducer anddirection of motion should be less than or equal to 60 degrees. Thisfigure shows that Color Doppler may also be applied for imaging the vasdeferens to distinguish the tube from the artery. This is aprecautionary method that the physician can utilize to prevent him orher from injecting the polymer material into a blood vessel.

In axial mode imaging using a high frequency probe (L15-7io), as perFIG. 8, the physician is able to discern the vas lumen (on the left handside) as opposed to the arteries and veins (on the right hand side). Thevas can be distinguished by the extensive smooth muscle layers thatsurround it. Around 90% of the vas deferens is made of smooth muscle. Inthis view, the physician can confirm that he or she successfullyisolated the vas deferens using the three-finger technique from thearteries and veins in the spermatic cord.

FIG. 9 is another axial ultrasound image of a patient's vas deferenslumen using a high frequency probe. Dr. Smith determined that the innerlumen diameter was approximately 1.41 mm in this patient.

FIG. 10 is a longitudinal ultrasound image performed in the samepatient. In this image, the inner diameter is shown to be approximately1.04 mm (much smaller than the other patient's) and the length of thevas visible is 1.73 cm. A hollow segment of the artery is also visibleunder the vas deferens. The vas can easily be distinguished by its largehollow lumen and large segment of smooth muscle.

Discussion:

This experiment tested and proved the feasibility of using a clamp andultrasound to observe the lumen of the vas deferens with highresolution. It took Dr. Smith only ten seconds to locate the lumen. Hewas confident that the ultrasound guidance would allow for the injectionto be performed percutaneously. This VASINTOMY™ procedure canpotentially reduce the surgical complications associated with avasectomy such as infection, granulomas, hematomas, and post-vasectomypain syndrome. The VASINTOMY™ is much less invasive and quicker than avasectomy.

If the urologist is injecting a solution into the lumen to reverse thecontraceptive gel, whether by dissolving or flushing it out, theprocedure can also be performed through the ultrasound-guided,percutaneous injection. Therefore, the VASINTOMY™ reversal will be muchquicker, simpler, and less invasive than the vasectomy reversal.

Example 2: Ultrasound Imaging of Synthetic Vas Deferens

This experiment involved ultrasound imaging synthetic vas deferens(purchased from SynDaver Labs (Tampa, Fla.)), which are commonly usedfor practicing vasectomy and vasovasostomy procedures. The synthetictissue has been validated to have similar mechanical properties to humanvas tissue. Thus, the synthetic vas deferens makes for a good ex vivomodel for the VASINTOMY™ procedure.

The synthetic vas deferens in FIG. 11 has an empty lumen while thesynthetic vas deferens in FIG. 12 contains an EVOH 32-15% polymer gelimplant. Both synthetic vas were held in place in Knox gelatin, whichwas formulated according to the manufacturer's recommendations forwater: powder ratio. Each vas was imaged in “general” focus and “high”frequency settings using an EZONO 4000 ultrasound. In the longitudinalimage of FIG. 11, the hollow lumen of the synthetic vas is clearlyvisible, while in the longitudinal image of FIG. 12, the lumen of thevas is filled with the polymer gel. Thus, the polymer gel successfullyoccluded the lumen. This figure also depicts that the EVOH polymer gelis highly echogenic on ultrasound without the need for echogenicparticles to be added.

The synthetic vas was also imaged in axial mode (FIG. 13A). In thismode, the hollow lumen is also visible (shown as the black void). Agraph of the grayscale values is shown in FIG. 13B. This graph depictsthat at the lumen, the grayscale value drops to below 50.

The synthetic vas containing EVOH 32-15% was also imaged in axial mode(FIG. 14A). In this mode, the hollow lumen is not visible due to thefact that the polymer gel completely occluded the lumen. A graph of thegrayscale values is shown in FIG. 14B. This graph depicts that thegrayscale value does not drop to below 50, suggesting that the lumen isnot void.

Example 3: Testing of a Vas-Occlusive Polymer in Male Rats

This testing relates to the development of a polymer gel for injectioninto the vas deferens of male rats for use as a contraceptive. Theproduct can be formulated for use in non-hormonal pet and humancontraception applications. A polymer is dissolved in sulfoxide (DMSO).Once injected, the DMSO is absorbed by the epithelial lining of the vasdeferens, resulting in precipitation of the polymer to form anocclusion. The gel formulation also contains microbubbles, which enhancethe contrast of the gel and allow the contraceptive to be visible underultrasound.

In embodiments, the product is a non-hormonal, ultrasound-imaginablecontraception for male pets and humans. Rats can be used to collectbaseline data on the effectiveness of the gel composition as acontraceptive. In such research, in vivo experiments should be usedbecause an in vitro model cannot accurately replace the anatomy andphysiology of a vas deferens by transporting and pumping sperm with thesame fluid dynamics. A mouse model cannot be used, because the vasdeferens is too small to manipulate using the procedure that is proposedfor pets and humans. Vasectomy studies are often conducted in rats (seeFlickinger C J. Alterations in the fine structure of the rat epididymisafter vasectomy. Anat Rec, 1972. 173(3): 377-300; Flickinger CJ.Ultrastructure of the rat testis after vasectomy. Anat Rec, 1972.174(4): 477-493; and Flickinger C J, et al. The influence ofvasovasostomy on testicular alterations after vasectomy in lewis rats.Anat Rec, 1987. 217(2): 137-145) due to their larger vas deferens andsimilar anatomy to larger mammals. A rat model is also the easiestmethod to study the effects of the gel on sperm concentration,viability, and motility as they pass through. Furthermore, a live animalmodel must be used to examine the histopathological findings monthsafter the gel has been implanted into the vas deferens.

Rats can be used to confirm the efficacy of an echogenic polymer gel asan ultrasound-imaginable, effective contraceptive. The echogenic gel canbe formulated as a polymer in DMSO with microbubbles, for example,homogeneously dispersed within. For example, the testing can beorganized such that four rats can receive an injection of purely DMSObilaterally as a negative control and four rats can receive a bilateralvasectomy as a positive control. As in the Koul study, twenty rats canundergo a bilateral injection of a non-echogenic polymer solution inDMSO and twenty rats can undergo a bilateral injection of an echogenicpolymer composition (see Koul V., et al. Reversibility with sodiumbicarbonate of styrene maleic anhydride, an intravasal injectablecontraceptive, in male rats. Contraception, 1998. 58(40): 227-231). Thevolume injected can be held constant at 60 μL and the concentration canbe 0.5 mg of polymer to 1 μL of DMSO. The sperm plug post-mating can beanalyzed once a week until 1 month, and then once every 30 days. In boththe echogenic and non-echogenic gel receiving groups, eight of thetwenty rats can be euthanized after 3 months to perform histopathologyof the vas deferens. Eight can undergo reversal of the contraceptive byflushing it out with 10% sodium bicarbonate at pH 8.9. One month afterreversal, the rats can be euthanized to examine their histology.Therefore, the total number of male rats necessary for such a study is4+4+20+20=48 rats. If mating of the male rats with female rats tocollect the sperm plug is performed, 12 female rats will be needed aswell. The total number of rats is 48+12=60 rats. 15 additional male ratscan be used for training purposes. Final count: 75 rats.

One month prior to the procedure, male rats can be allowed to mate withfemale rats to determine their mating performance. Only male rats with aproven mating performance should be used. Should the female rats becomepregnant, the rat pups can be euthanized, and the females can be allowedto recover and used for the subsequent mating studies. For theprocedure, the male rat is weighed and anesthetized by intraperitonealinjection with Ketamine/Xylazine (80/10 mg/kg) combined dose (the sameanesthesia can be used as maintenance anesthesia during surgery if theprocedure lasts more than 30 minutes). Sustained release buprenorphine(0.5 mg/kg) is injected subcutaneously into the scruff of the neck as aprimary analgesic. The rats' eyes are treated with petrolatum ophthalmicointment to prevent dry eyes. Once unconscious, the rats are checked forthe absence of a rear foot reflex by pinching the toe, and for theabsence of palpebral reflex. Surgeries are performed under sterileconditions, and follow the ACUC Policy on Rodent Survival Surgery. Thevasectomy is performed through a vertical midline transabdominalapproach. The anesthetized male rat is placed on its back to expose theabdomen. The fur is removed from the ventral area above the penis usingelectric clippers. The shaved area is sanitized by wiping threealternating times with 10% povidone iodine and 70% ethanol. The steriledrape with a hole exposing the shaved area is placed on the rat. A 10-15mm longitudinal skin incision is made in the medial line of the abdomen,about 1 cm above the penis. Then, a 5-10 mm longitudinal incision ismade in the linea alba. The testicular adipose pad is pulled withdissecting serrated forceps to expose the testis, vas deferens, andepididymis. The vas deferens is located medial to the testis and is aclearly distinguishable free tube, unconnected to the testis, and has ablood vessel running along one side.

In order to perform a vasectomy, the vas deferens loop is held withforceps. At the same time, another pair of forceps is heated with aBunsen burner until it turns red. Then, the vas is cut and cauterized intwo points at once with the hot forceps. The cut should be 5 mm of thevas deferens and leave two clearly separated ends. The testicle,epididymis, and vas deferens are moved back to the abdominal cavity.This procedure is repeated on the contralateral side through the sameincision in the abdomen. Rats that undergo a bilateral implantation ofthe gel can have the same procedure done, as the rats receiving avasectomy, except instead of cutting/cauterizing the tube, the lumen ofthe vas is injected with 60 μL of the polymer/microbubble/DMSOcomposition. The injection is done slowly (0.05 cc/min) using a 23 gaugeneedle with the flow directed towards the ampulla. The polymercomposition does not require sterilization because DMSO is an organicsolvent and does not have any bacteria in it. However, sterilization ofthe composition can be performed according to any available technique,including, for example, by using a 0.22 micron filter that is DMSOcompatible, dry heat, autoclave, ethylene oxide, gamma, and/or e-beam,etc. Compression can be maintained with the fingers, just distal to theinjection site, to avoid retrograde flow. The polymer precipitates intoa gel within a few minutes, causing partial occlusion of the vas. Thetesticle, epididymis, and vas deferens is moved back into the abdominalcavity. The procedure is repeated on the contralateral side. The musclewill be sutured with one or two horizontal mattress stitches made with5-0 absorbable sutures (Dexon), and the skin is sutured with anon-absorbable 4-0 monofilament suture (Prolene). Primary post-operativeanalgesics and a fluid bolus (5-10 mL) are given at this point. The ratis placed on a warm stage and allowed to recover from anesthesia(conscious and maintain sternal recumbency). Ketoprofen (2.5 mg/kg) isgiven subcutaneously as a secondary analgesic to provide systemicanalgesia after the procedure. The rats are monitored every day for 3-4days post-operation to ensure no surgery related complications such asbleeding, etc. arises. The skin sutures are removed after 10-14 days.Afterward, they are monitored once weekly to ensure the animals areproperly recovering and not demonstrating any signs of discomfort orpain. Once fully recovered, the male rat is allowed to mate with afemale rat. Mating is confirmed by the presence of sperm plugs (vaginalplugs). It is hypothesized that the contraceptive will preventconception, but should this happen, the rat pups that are born will beeuthanized with carbon dioxide followed by decapitation. The female ratsare allowed to recover and be used for subsequent mating. The male alsoundergo non-invasive ultrasound imaging twice a month for the presenceof the polymer gel in the vas deferens. For this, the rat is sedatedusing an intraperitoneal injection of Ketamine (60 mg/kg) and Xylazine(3.0 mg/kg). The ultrasound probe that can be used is the Acuson 15L8-Sat a frequency of 8 MHz. The duration should not be longer than 25minutes. At 3 months, eight of the rats who received the polymer implantare euthanized by isoflurane overdose and cervical dislocation, andtheir tissues (distal vas, injection site, proximal vas, caudaepididymis, caput epididymis, and testis) are harvested and examined forthe presence of the polymer gel. The inflammatory response of the walland adventitia of the vas deferens is studied. Eight of the rats undergoreversal of the polymer through a similar procedure as described above,except that a 0.5 mL solution of 10% sodium bicarbonate at pH 8.4 isinjected into the vas to flush out the gel. The time to regain fertilitycan be measured. One month later, the rats who received a reversal areeuthanized and histology of the genitourinary tissues is performed.

The rats are observed every day for 3-4 days post operation and weighedonce weekly to determine if any problems (redness, swelling, infection)or distress (weight loss >20%, abdominal breathing, lethargy, twitching)appear. If there are any problems, additional analgesics (Buprenorphine,Ketoprofen) are administered for 1-2 days to see if signs are relieved.

Example 4: Method for Forming the Polymer Solutions

In this example, the inventive composition consists of a polymer,ethylene vinyl alcohol (EVOH), dissolved in an organic solvent, such asdimethylsulfoxide (DMSO). EVOH pellets are weighed and suspended in99.9% DMSO at a desired weight percentage using the following formula:

Wt %=(mass of EVOH)/[(mass of EVOH)+(mass of DMSO)]

The equation can also be rearranged to calculate for the mass of EVOHneeded to achieve a certain weight %:

Mass of EVOH=[(wt %)(mass of DMSO)]/(1−wt %)

Once the EVOH pellets are weighed out and suspended in DMSO, thesolution is vortexed and left on an orbital shaker for >6 hours(preferably overnight) at >50 degrees C.

The molecular structure of EVOH is shown in FIG. 15B. The structure ofstyrene maleic acid (SMA) is shown in FIG. 15A for comparison.

It has been shown that the optimal concentrations for the compositionare between 6 and 20 wt % of EVOH, with this range seemingly fallingwithin the concentrations ranging from 10-18 wt %. EVOH begins to reachits saturation point at 20 wt %. As the wt % increases, so does theviscosity making injection of the material difficult.

The molecular weights and monomer ratios of EVOH may also be controlledduring the polymer synthesis process. For instance, EVOH 27 signifiesthat the pellet has an ethylene content of 27% and vinyl alcohol contentof 73%. For vas-occlusive purposes, the present inventors shown that theoptimal ethylene contents are from 27% to 38%, preferably 31-33%. Theethylene content controls the hydrophilicity/hydrophobicity of thepolymer: the lower the ethylene content, the higher the vinyl alcoholand therefore, greater hydrophilicity. These factors can impact how thehydrogel forms in water, swells, absorbs water, its viscosity, thermalstability, and durability.

The hydrogen bonds present in the skeletal structure of EVOH polymer,and forming at its crosslink locations, allow for a high degree ofmechanical strength. The selection of specific molecular weight(corresponding to the length of the carbon chain backbone) of EVOHallows for the specific observable elastic properties we see in theinventive compositions. Shorter, lower molecular weight polymers havebeen observed to exhibit fracture and for lack of a better word“brittleness,” while the higher molecular weight formulations exhibithigher elastic properties.

Higher molecular weight compounds (corresponding to longer polymerchains) allow for increased polymer flexibility. This is due to the factthat larger molecular weight chains allow for rotation of the chainaround the single carbon-carbon bond.

In embodiments, the polymers can have a weight average molecular weight(M_(w)) or number-average molecular weight (M_(n)) ranging from about1,000 to 1,000,000 as measured by GPC (gel permeation chromatography)with polystyrene equivalents, mass spectrometry, or other appropriatemethods. In embodiments, the number-average molecular weight (M_(n)) orthe weight average molecular weight (M_(w)) of polymers of the inventioncan range from about 1,000 to about 1,000,000 Daltons, such as fromabout 3,000 to about 60,000 Daltons, or from about 20,000 to about90,000 Daltons, or from about 150,000 to about 900,000 Daltons, or fromabout 200,000 to about 750,000 Daltons, or from about 250,000 to about400,000 Daltons, or from about 300,000 to about 800,000 Daltons, and soon. Further, the degree of polymerization of the polymers in embodimentscan range from 1 to 10,000, such as from 50 to 500, or from 500 to5,000, or from 1,000 to 3,000.

The chain length or degree of polymerization (DP) can have an effect onthe properties of the polymers. In the context of this specification,the degree of polymerization is the number of repeating units in thepolymer molecule. Included are polymers comprising from 2 to about10,000 repeating units. Preferred are polymers comprising from 5 to10,000 repeating units, such as from 10 to 8,000, or from 15 to 7,000,or from 20 to 6,000, or from 25 to 4,000, or from 30 to 3,000, or from50 to 1,000, or from 75 to 500, or from 80 to 650, or from 95 to 1,200,or from 250 to 2,000, or from 350 to 2,700, or from 400 to 2,200, orfrom 90 to 300, or from 100 to 200, or from 40 to 450, or from 35 to750, or from 60 to 1,500, or from 70 to 2,500, or from 110 to 3,500, orfrom 150 to 2,700, or from 2,800 to 5,000, and so on.

Example 5: Manufacture of Microbubbles

Custom-synthesized polystyrene microbubbles were manufactured. Amicroscopic image of the microbubbles is shown in FIG. 16.

Example 6: Biocompatibility

The cytotoxicity of various polymer formulations including ethylenevinyl alcohol (EVOH), styrene maleic acid (SMA), and control (no polymergel) was evaluated on mouse Leydig cells (testosterone-producingendocrine cells found in the testes). Leydig cells were chosen as a“worst-case scenario” using a cell line that is unique to the malereproductive tract, and which is sensitive to noxious agents. The assayutilized was an MTT assay, a widely accepted assay for measuring theeffect of drugs and devices on cellular cytotoxicity. The polymer gelformulations were allowed to incubate with the cells for 24 hours. Afterthe addition of the MTT reagents and following the standard MTTprotocol, the absorbance was read at 570 nm. The results are shown inFIG. 18. Increasing absorbance levels reflect greater mitochondrialactivity and therefore, biocompatibility. EVOH polymer formulationscytotoxicity in this assay was compared to that of SMA polymerformulations and the negative control (no gel); the results are shown inFIG. 19. Comparing cytotoxicity of EVOH and the negative control usingANOVA testing and post hoc Tukey testing showed a p-value of 0.076(greater than an alpha of 0.01), suggesting that there is no significantstatistical difference in cytotoxicity between EVOH polymer formulationsand the control. Similar comparisons were performed to assess therelative cytotoxicity of SMA to the control; in this case, the resultingp-value was 0.001 (<0.01), suggesting a significant statisticaldifference between the two exposures. Therefore, it can be concludedthat the EVOH polymer formulations tested provide significantly higherbiocompatibility compared to SMA polymer formulations. It was alsoobserved that the DMEM media of the cells exposed to SMA polymer gelsturned from pink to yellow, suggesting a pH change from neutral toacidic. It is hypothesized that the maleic acid moieties of SMA increasethe hydrogen ion concentration, resulting in the pH difference andleading to cell death.

FIG. 20 shows the results from an MTT assay used to assess thecytotoxicity of a variety of EVOH polymer formulations comprisingdifferent monomer ratios of ethylene: vinyl alcohol and weightpercentages, as well as several SMA polymer formulations of constantmolecular weight (350,000 daltons), but different weight percentages(15%, 20%, and 25%). The VASALGEL formulation is SMA, 350 kD, and 25 wt% in DMSO, which has shown to be very cytotoxic. Similar to the previousexperiment, all EVOH formulations were more biocompatible than SMA. Thisdata was analyzed using ANOVA testing and several post-hoc Tukeytestings to draw conclusions on the comparisons.

In FIG. 21, post-hoc Tukey testing was used to assess the effect ofmonomer ratios on cytotoxicity. EVOH 27 polymers are more cytotoxic thanEVOH 32, EVOH 38, and the negative control (p values of 0.029, 0.003,and 0.002 respectively). Similarly, it can be concluded that thecytotoxicity of EVOH 32 and EVOH 38 are no different than that of thenegative control (p values of 0.248 and 0.499 respectively). Therefore,for vas-occlusion, EVOH 27 should be eliminated as a polymer candidate.

In FIG. 22, post-hoc Tukey testing was used to assess the effect ofpolymer weight percent on cytotoxicity. Conclusions from these resultsare that EVOH at 5 wt % has a significantly higher cytotoxicity whencompared to the other wt % tested and the negative control (p values of0.001, 0.001, 0.002, and 0.001 for mass weight percents 10%, 15%, 20%,and the negative control respectively). Therefore, EVOH 5 wt % should beeliminated. This is non-obvious because ONYX and URYX, two medicaldevices FDA approved for embolization, use 6 wt %. This wt % is notconcentrated enough for effective and safe vas-occlusion.

FIG. 23 shows the results of non-GLP Cytotoxicity (MEM Elution) forvarious polymer solutions. EVOH 32-10%, EVOH 32-15%, and EVOH 28-15% didnot exhibit toxicity in the assay.

Several conclusions can be drawn as a whole from the cytotoxicitytesting described above. First, SMA based polymers present withsignificantly higher cytotoxic effects when compared to EVOH polymers.Second, low weight percent EVOH polymers are more cytotoxic than high wt% formulations. This is likely due to the lower degree of polymerizationthat occurs with these samples, and thus the presence of more activemonomer units that can have negative cellular effects. Finally, EVOH 27formulations of all weight percentages demonstrate increasedcytotoxicity when compared to the control, suggesting that suchformulations should be excluded as viable products.

Example 7: Rodent Animal Model

Sexually mature, 11-16 week old rats received either vasectomy orVASINTOMY™ via surgical approach transabdominally. The techniqueinvolved injection of 60 μL of SMA or EVOH gel approximately 4 cm caudalto the prostate with injection proceeding antegrade. The animalsrecovered for a period of 3 days or 14 days. At the end of the timeperiod, the rats were humanely euthanized and their reproductive tractwas studied by macroscopic and microscopic pathologic examinations.

FIG. 24 below shows the results of a VASINTOMY™ in an animal model witha detailed view of the injected EVOH 32-15%. Note the yellowish-white,opaque material visualized through the outer muscular tunics of the vasdeferens.

In preliminary rodent trials, the present inventors' studies showed thatSMA incited an intense inflammatory reaction within the muscular wall orspermatic cord connective tissues. FIGS. 25A-25D show histopathologyimages of the vas deferens of 12 week old, Sprague-Dawley male ratsreceived bilateral vasectomy or VASINTOMY™ using SMA. Vas deferens wereexamined via histopathology on post-surgical day 14 after implantation.As shown in the figures, sections of vas deferens which containedsurgically implanted SMA gels were characterized by presence of numerousinflammatory cells. The cells were associated with abundant intra- andextra-cellular amorphous, lightly basophilic material and/orspermatozoa. The material was noted to be PAS positive on specialstains, and was consistent with SMA hydrogel. Many of these collectionsand nodules were present in the wall of the vas deferens or adjacent fator fascia. These findings indicate that further study is needed tocharacterize SMA in mammalian animal model systems, and that EVOH mayprovide a better composition.

Studies by the inventors have demonstrated that EVOH gel injection inrodent animal models induces a dense plug that has the potential tocompletely occlude the vas deferens lumen resulting in azoospermia. Inone preliminary rodent trial, 16-week old, Sprague-Dawley male ratsreceived bilateral vasectomy or VASINTOMY™ using EVOH 32-15%. Vasdeferens were examined via histopathology on post-surgical day 3 afterimplantation. EVOH was associated with inflammation in some animals,however, this reaction was different from that of SMA, in that there wasrapid connective tissue proliferation associated with the vasalepithelial and lamina proprial layers. The changes, including fibrosisand fibroplasia were similar to microscopic changes evokedintravascularly with FDA-approved use of the composition in arteries.Further characterization of the in vivo safety and efficacy areforthcoming.

The following figures show histology images which represent the changein the luminal diameter and content of the vas deferens of a 16 week oldrodent subjects on post-surgical day 3. FIG. 26A shows an age-matchedcontrol vas deferens (day 0). Note the small diameter, of the lumenrelative to the thick muscular wall of the tunica muscularis. FIG. 26Bshows the vas deferens (HE staining) of a treated subject. EVOH 32-15%in lumen of vas deferens is associated with dilation of lumen andmuscular tunics, with retention of overall diameter of the vas deferenswhen compared with control. FIG. 26C (PAS staining) shows that the EVOH32-15% serial section of FIG. 26B exhibits intense affinity of thehydrogel (polymer implant) for PAS stain.

FIGS. 27A-27D are histology images which show that the resting diameterof the epithelial lamina propria is approximately 200 μm. The vasluminal diameter with polymer implant increased to nearly 1.0 mm in thissubject. Dilation of the lumen is characterized by attenuation andcompression of the normal mucosal folds with compression of the smoothmuscle tunic cells, while preserving overall total vas deferensdiameter.

Example 8: Injection Volume for Humans Based on Vas Diameter

FIG. 28 is a table showing the relationship between inner diameter(dilated) and injection volume and for various targeted occlusion sizesfor human vas-occlusive contraception. The source of the table is astudy by Zhao Shengcai.

Example 9: Porosity

Porosity is an essential parameter to predict the efficacy of theinventive compositions. The polymer gel must have porosity tailored forpreventing the passage of sperm cells, while allowing for appropriatelevels of fluid infiltration. Sperm cells present with a wedge-shapedhead with dimensions of 3 μm×5 μm. Thus, the vas-occlusive contraceptiveshould have an average pore size below 3 microns.

Porosity of the gels was assessed several ways. Scanning electronmicroscopy (SEM) allowed for visualization of the pores on a 10 μmscale. Pore size was assessed by direct software-assisted measurement.However, this method was not optimal for determining the average of allthe pores on the polymer's surface. Brunauer-Emmett-Teller (BET)analysis is a superior quantitative method which can measure the numberof gas molecules that adhere to a surface at a given pressuredifferential. This number can be related to the surface area of thesample in question via BET analysis. BET surface area analysis andsubsequent Barrett-Joyner-Halenda (BJH) pore size and volume analysisallow for the comparable quantification of sample pore size and surfacearea. The BET results for select polymer candidates are shown in thetable of FIG. 29, while representative SEM images are shown in FIGS.30A-C. The SEM results show that EVOH 32-10% (FIG. 30A) did have smallpores present, while EVOH-32 15% (FIG. 30B) and 20% (FIG. 30C) did not.The 15% had a very flat surface while 20% exhibited classic spinoidaldecomposition (meaning that there was a phase separation of the polymerfrom solvent). Basically, 20% was the saturation point for EVOH.

Pore size was determined using the following equation:

Average Pore Diameter=(4*Av. Pore Volume)/Specific Area

BET analysis suggests that EVOH polymers, in general, has a much loweraverage pore diameter than SMA polymers. This supports the fact thatEVOH films are used for wrapping and preserving food, to prevent gasesfrom traveling through. Furthermore, as the weight % increased, thepores on the EVOH gels became smaller. At EVOH 32-15 wt % and 20%, theaverage pore size is below the size of atoms (0.5 nm). Initialexperiments suggest that at high wt %, EVOH is essentially non-porous.

Pore size is an essential modifiable parameter relating to the efficacyof the inventive compositions as a vas-occluding technology. Theporosity of male-contraceptive compositions must be tailored toappropriately allow flow through of fluid, while preventing suchnegative effects as sperm granuloma, vas deferens rupture, andsubsequent epididymitis.

Example 10: Hardness/Elasticity

In a study by Bank et al., (see Bank et al., “Contribution of Collagen,Elastin, and Smooth Muscle to In Vivo Human Brachial Artery Wall Stressand Elastic Modulus”, Circulation. 1996; 94:3263-3270), it wasdetermined that the greatest active stress generated by the smoothmuscle in the brachial artery was 1.24×10̂6 dynes/cm². If it is assumedthat 70% of the brachial artery cross-sectional area is smooth muscle,then the active stress generated by the smooth muscle is 1.77×10̂6dynes/cm² (or 177 kPa).

Elastic modulus is representative of a material's ability to deformunder stress without undergoing permanent plastic deformation. Thepresent inventors have predicted the stress that the vas deferens willbe generate during ejaculation based on the thickness of smooth musclein the organ and the known force-production properties of smooth muscleduring arterial contraction (see reference above). Therefore, using thisprediction of the stress that the inventive compositions will be under,we are able to determine a design parameter defining the elasticproperties that our compositions must have.

Assuming the smooth muscle in the vas has similar elastic properties asthe smooth muscle in the brachial artery (124 kPa) and 90-93% of thecross-sectional area of the vas is made of smooth muscle (this wascalculated using measurements of inner and outer vas diameters), thenthe maximum stress that the smooth muscle in the vas can generate is1.37×10̂6 dynes/cm̂2 or 137 kPa. As per the table in FIG. 31 and the graphin FIG. 32, only EVOH 32-20% and SMA-25% would be able to withstand thestress of the vas since their elastic modulus is greater than 137 kPa.

Example 11: Spermicidal Effects of the Compositions

In addition to blocking sperm, it was also discovered that differentpolymer formulations may have spermicidal characteristics. This effect,most likely a secondary “mode of action”, may help increase the efficacyof the inventive compositions in vivo. An in vitro set-up was designedwhere the gels were precipitated on top of a mesh in cell strainers andhuman sperm samples were added to the top of the gels. After 30 minutesof exposure, sperm cells were observed under a microscope and scored formotility. The results are shown in FIG. 33. Initial ANOVA analysis ofthe results showed a significant difference between treatment groups,therefore post-hoc Tukey HSD was applied in order to determine whetherEVOH and/or SMA polymers decreased sperm motility (FIG. 34A). Tukeytesting (FIG. 34B) showed that sperm interaction with EVOH and SMApolymers significantly decreased motility below WHO standards whencompared with DMSO negative control. The decrease in motility in theDMSO (negative control) samples were most likely due to naturalphysiological changes in motility over time and were likely not causedby DMSO itself. Furthermore, EVOH 32, EVOH 38, and SMA did a better jobof decreasing sperm motility than EVOH 32.

Spermicidal characteristics of the EVOH polymer implant was tested byusing a viability assay in which human sperm cells were incubated withgels formed on cell strainers. After 30 minutes of exposure, sperm cellswere stained with Trypan Blue dye and observed under a microscope. Thepercentage of viable sperm were recorded for each sample. The resultsare shown in FIG. 35. Initial ANOVA analysis of the results showed asignificant difference between treatment groups, therefore post-hocTukey HSD was applied in order to determine whether EVOH and SMApolymers decreased sperm motility (FIG. 36A). Tukey testing showed thatincubation with EVOH polymers significantly decreased motility whencompared with DMSO negative control (FIG. 36B). The significant decreasein viability due to incubation with DMSO was most likely due to naturalphysiological changes in viability and not likely caused by DMSO itself.

Example 12: Viscosity and Rheological Properties of the PolymerSolutions

A controlled shear rate test was performed with an Anton Parr rheometer(Physica MCR 301) to measure the viscosities of SMA (350 kD) 22 wt % andEVOH 32-15%. The viscosity as a function of shear rate graph shown inFIG. 37 shows that SMA has an extremely pronounced shear-thinningbehavior, meaning that its viscosity decreases as the shear rateincreases. This behavior stems from the increase in the disentanglementof polymer molecules at higher shear rates. However, the graph showsthat the viscosity for EVOH is independent of shear rates. ThisNewtonian fluid behavior is particularly significant in that it is thesimplest mathematical model of fluids that account for viscosity, andthe application of Hagen-Poiseuille relation that relates the viscositymeasurements to the formulation of injection mechanisms is much moresimplified compared to one for the non-Newtonian shear-thinning behaviorexhibited by SMA. The graph also illustrates that the viscosity of SMAat 22 wt % is about 4 times higher than that of EVOH 15 wt %. Therelatively small value of viscosity for EVOH is also allows for moreefficient injection mechanism, as the smaller needle gauge (with largerbore) will be required to extrude the polymer solution for higherviscosity solutions.

The effect of ethylene mol % on the viscosity of EVOH was tested with acontrolled shear rate test under isothermal condition (T=37 degreesCelsius). The graph in FIG. 38 shows the lower shear rate range of thedata for EVOH. The results show that an increase in the ethylene mol %does not necessarily correspond to an increase in the viscosity. It isgenerally established that higher molecular weight of polymers resultsin increased strength, stiffness, and therefore viscosity. Based on thisgeneral premise and the viscosity data, it is hypothesized that the EVOH38 has a lower molecular weight than EVOH 27. To prove this hypothesis,GPC (Gel Permeation Chromatography) or DSC (Differential Scanningcalorimetry) will need to be conducted to accurately determine themolecular weight distribution.

To determine the gel time in form of the sol/gel transition point, anoscillatory test in the linear viscoelastic (LVE) deformation range wasperformed for EVOH 32 at different wt %. The results are shown in FIG.39. The melting point occurs at the crossover point of storage modulusG′ and loss modulus G″. For 10 wt %, the melting point occurred atapproximately 144 degrees Celsius. The glass transition point occurs atthe first inflection point of G′, which was absent in this run. Moretrials are needed to confirm the accuracy of the data.

For EVOH 32-15% (FIG. 40), the melting point occurred at 149 degreesCelsius, which is 5 degrees higher than that for 10 wt %. The glasstransition point occurred at 102 degrees Celsius, indicating that thereversible transition from a hard, brittle material to a soft, rubberymaterial occurs at that temperature.

For EVOH 38-15% (FIG. 41), the melting temperature occurred at 116degrees Celsius and the glass transition temperature occurred at 87degrees Celsius. Compared to EVOH 32-15%, the melting point temperaturedecreased by 33 degrees Celsius and the glass transition temperature by15 degrees Celsius. This comparison leads to the conclusion that forhigher ethylene mol % of EVOH, the transition of the polymer solutionfrom a solid state to a liquid/gel-like state occurs at a lowertemperature.

Example 13: Hydrogel Swelling Studies

FIG. 42 represents the mass percent swelling of different polymerformulations in a proprietary media (hereby known as IVVM) simulatingthe human vas deferens environment by having the same osmolarity and pH.Mass percent swelling for each sample was measured by freeze-drying tensamples of cured polymer formed from a volume of 0.1 mL of resinouspolymer, weighing these samples to determine an initial mass, exposingeach of the samples to 1 mL of IVVM for three hours, and then reweighingto determine the final mass after swelling. Percent swelling wascalculated as: % SW=((Mf−Mi)/Mi)*100. The results of this experimentsuggest that percent swelling decreases as the weight percent of thepolymer increases and that EVOH 38 has a higher percent mass swellingthan EVOH 32. The fluid infiltration of EVOH 32-10% and EVOH 32-15% areshown in the graphs of FIG. 43.

Example 14: Degradation Studies

FIG. 44 is a summary of the results of an accelerated aging study toassess the stability/durability of several different EVOH polymerformulations in vitro. The experiment has been designed to simulate thevas deferens micro-environment, which involves maintaining the hydrogelsat the same temperature found in the vas deferens (35 degrees Celsius;slightly below body temperature) and in media consisting of the sameosmolarity (ion concentrations) and pH. A proprietary media, coined invitro vas media (IVVM), was used to simulate this environment. Sampleswere introduced to 1 mL of IVVM after being lyophilized (freeze-dried).Initial swelling has been observed for several formulations as thesamples are re-hydrated. Degradation is being measured by weight % gainor loss over time.

Polymer gels with higher stability in a biological environment will showa slower rate of degradation (or mass % lost) when compared to other,degradable polymers. The results of this study demonstrate that all EVOHhydrogels are non-degradable for 1 month. The EVOH hydrogels swell toalmost double their weight in the first two days. Their mass thenremains steady for the remaining weeks. The results hold similar to theswelling study, where EVOH 38 has been shown to swell more than EVOH 32.However, in this degradation study, the 15 wt % EVOH gels swelled morethan the 10 wt %.

Unlike EVOH, SMA was highly degradable (FIG. 45). The polymer swelledfor 6-10 days (depending on the wt %) and then began to degrade at 35degrees C. and in vas-simulating media. It took 20-25 days for the SMApolymers to degrade. Thus, it is shown that SMA would not be a goodcandidate for long-term (>1 year) vas-occlusive contraception.

Example 15: Echogenicity

Formulations of ninety-six test gels were created within a 96-well plateand arranged in a Knox gelatin construct to compare the averageechogenicity of the different compositions. Each well was filled with0.5 mL of gel solution and was then exposed to 1.5 mL of DI water. Themixture was given a quick stir to ensure that the gel solutions had fullcontact with water and were allowed to precipitate and harden. The Knoxgelatin was created according to the manufacturer recommendedwater-powder ratio. Eight technical replicates were performed for eachof the twelve formulations, and each gel was imaged in five differentlocations throughout their length to average out non-homogenoussections. The gels were imaged with an Ezono 4000 ultrasound using alinear transducer in “general” focus and “high” frequency settings.

FIG. 46 shows axial cross-sections of representative gels. Thebrightness of these gels relative to the background would be the metricfor how ultrasound visible they would be within the body during theVASINTOMY™ procedure. FIGS. 47A-47C show three of the EVOH 32 gels in anexpanded view.

The entirety of the gel within each image was traced out on ImageJ andan average pixel brightness of that image was recorded. This value wouldbe averaged with the other four images from that gel to ensure arepresentative brightness value. This process was repeated for eachreplicate and an average brightness and standard deviation for each gelformulation was generated. The results are shown in the graph in FIG.48.

From this data, it can be seen that EVOH 27 was more echogenic than EVOH32 and EVOH 38. There was not a clear pattern in increase inechogenicity based on weight %. All gels were strongly contrasted withthe background (>35 brightness) and would easily be distinguished fromthe lumen of the vas using ultrasound.

Example 16: Ultrasound Imaging of Ethylene Vinyl Alcohol Gels with andwithout Microbubbles

Gels were precipitated in a cuvette filled with water-based ultrasoundgel. The gels were 10% EVOH (32% ethylene content) in DMSO. Full-widthhalf-maximum resolution was calculated for images taken. Lateral andaxial resolution of imaged gels from varied double syringe agitationprotocols (1 week trial). Double syringe agitation was performed usingEVOH 32-10% by varying the # of pumps (40 or 80) and volume of airintroduced (0.25 mL or 0.75 mL). The gels were precipitated in a cuvettewith ultrasound goo and the full-width half-maximum resolution wascalculated for the ultrasound images taken. Lateral (FIG. 17A) and Axial(FIG. 17B) resolutions of various echogenic EVOH gel images at Day 0 andDay 8 (n=1) are shown, with P=plunges and A=mL of air introduced todouble syringe system.

In general, the higher the resolution, the lower the echogenicity. Thus,the non-echogenic gels had lower longitudinal and axial echogenicitythan the gels containing air microbubbles formed by agitation.Furthermore, the echogenicity increased with increased # of pumps. Theamount of air introduced did not significantly increase theechogenicity. In most cases, the polymer lost echogenicity after 8 daysin the cuvette. However, the echogenic gels formed with 80 pumpsincreased in echogenicity from day 0 to day 8.

The present invention has been described with reference to particularembodiments having various features. In light of the disclosure providedabove, it will be apparent to those skilled in the art that variousmodifications and variations can be made in the practice of the presentinvention without departing from the scope or spirit of the invention.One skilled in the art will recognize that the disclosed features may beused singularly, in any combination, or omitted based on therequirements and specifications of a given application or design. Whenan embodiment refers to “comprising” or “including” certain features, itis to be understood that the embodiments can alternatively “consist of”or “consist essentially of” any one or more of the features. Otherembodiments of the invention will be apparent to those skilled in theart from consideration of the specification and practice of theinvention.

It is noted in particular that where a range of values is provided inthis specification, each value between the upper and lower limits ofthat range is also specifically disclosed. The upper and lower limits ofthese smaller ranges may independently be included or excluded in therange as well. Further, the ranges of values disclosed in thisspecification are not limited to those explicitly disclosed but caninclude any value recited in this specification as an upper or lowerlimit. The singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. It is intended that thespecification and examples be considered as exemplary in nature and thatvariations that do not depart from the essence of the invention fallwithin the scope of the invention. Further, all of the references citedin this disclosure, including patents, published applications, andnon-patent literature are each individually incorporated by referenceherein in their entireties and as such are intended to provide anefficient way of supplementing the enabling disclosure of this inventionas well as provide background detailing the level of ordinary skill inthe art.

What is claimed is:
 1. A composition comprising: about 5 to about 20weight percent of an ethylene vinyl copolymer; wherein the ethylenevinyl copolymer is dissolved in a solvent; wherein the ethylene vinylcopolymer comprises about 27 to about 48 mole percent of ethylene. 2.The composition of claim 1 comprising about 10 to about 20 weightpercent of the ethylene vinyl copolymer.
 3. The composition of claim 1comprising about 31 to about 33 mole percent of ethylene.
 4. Thecomposition of claim 1, which has a viscosity in the range of about 1 to7 Pa*s.
 5. The composition of claim 4, which has a viscosity in therange of about 1-3 Pa*s.
 6. The composition of claim 1, wherein theethylene vinyl copolymer is ethylene vinyl alcohol (EVOH).
 7. Thecomposition of claim 1, wherein the solvent is an organic solvent. 8.The composition of claim 1 comprising one or more ultrasound contrastagents.
 9. The composition of claim 1 comprising one or moremicrobubbles.
 10. The composition of claim 9, wherein the microbubblesare present in the composition at a concentration between about 1×10² toabout 1×10⁹ microbubbles/ml.
 11. The composition of claim 9, wherein themicrobubbles have an average size in the range from about 1 to about1,000 μm in diameter.
 12. The composition of claim 9, wherein themicrobubbles have a shell encompassing a gas or a plurality of gaseschosen from one or more of air, nitrogen, argon, or perfluorocarbon. 13.The composition of claim 12, wherein the shell of the microbubblescomprises a polymer, a lipid, a protein, a surfactant, a monosaccharide,a polysaccharide, or glass.
 14. The composition of claim 13, wherein theshell of the microbubbles comprises a polymer comprising one or more ofnatural or synthetic monomers, polymers, copolymers or block copolymers,biocompatible monomers, polymers, copolymers or block copolymers,polystyrene, neoprene, polyetherether 10 ketone (PEEK), carbonreinforced PEEK, polyphenylene, PEKK, PAEK, polyphenylsulphone,polysulphone, PET, polyurethane, polyethylene, low-density polyethylene(LDPE), linear low-density polyethylene (LLDPE), high-densitypolyethylene (HDPE), polypropylene, polyetherketoneetherketoneketone(PEKEKK), nylon, TEFLON® TFE, polyethylene terephthalate (PETE), TEFLON®FEP, TEFLON® PFA, and/or polymethylpentene (PMP) styrene maleicanhydride, styrene maleic acid, polyurethane, silicone, polymethylmethacrylate, polyacrylonitrile, poly (carbonate-urethane), poly(vinylacetate), nitrocellulose, cellulose acetate, urethane,urethane/carbonate, polylactic acid, polyacrylamide (PAAM), poly(N-isopropylacrylamine) (PNIPAM), poly (vinylmethylether), poly(ethylene oxide), poly (ethyl (hydroxyethyl) cellulose), poly(2-ethyloxazoline), polylactide (PLA), polyglycolide (PGA),poly(lactide-co-glycolide) PLGA, poly(e-caprolactone), polydiaoxanone,polyanhydride, trimethylene carbonate, poly(β-hydroxybutyrate),poly(g-ethyl glutamate), poly(DTH-iminocarbonate), poly(bisphenol Aiminocarbonate), poly(orthoester) (POE), polycyanoacrylate (PCA),polyphosphazene, polyethyleneoxide (PEO), polyethylglycol (PEG),polyacrylacid (PAA), polyacrylonitrile (PAN), polyvinylacrylate (PVA),polyvinylpyrrolidone (PVP), polyglycolic lactic acid (PGLA),poly(2-hydroxypropyl methacrylamide) (pHPMAm), poly(vinyl alcohol)(PVOH), PEG diacrylate (PEGDA), poly(hydroxyethyl methacrylate) (pHEMA),N-isopropylacrylamide (NIPA), poly(vinyl alcohol) poly(acrylic acid)(PVOH-PAA), collagen, silk, fibrin, gelatin, hyaluron, cellulose,chitin, dextran, casein, albumin, ovalbumin, heparin sulfate, starch,agar, heparin, alginate, fibronectin, fibrin, keratin, pectin, elastin,ethylene vinyl acetate, polyethylene oxide, PEG or any of itsderivatives, PLLA, PDMS, PIPA, PEVA, PILA, PEG styrene, Teflon RFE,FLPE, Teflon FEP, methyl palmitate, NIPA, polycarbonate,polyethersulfone, polycaprolactone, polymethyl methacrylate,polyisobutylene, nitrocellulose, medical grade silicone, celluloseacetate, cellulose acetate butyrate, polyacrylonitrile, PLCL, and/orchitosan.
 15. The composition of claim 1 comprising an agent thatdecreases the fertility, motility, or viability of sperm cells.
 16. Thecomposition of claim 15, wherein the agent is one or more of a smallmolecule, protein, antibody, peptide, nucleic acid, or fragment thereof,or nonoxynol-9, oxtoxynol-9, benzalkonium chloride, or chlorhexidine.17. The composition of claim 1, which is a hydrogel formulated such thatwhen the hydrogel is exposed to water the hydrogel swells no more than80% by volume.
 18. The composition of claim 1, which is capable ofpolymerizing into a polymer and wherein the polymer is non-biodegradablein a human or animal body or begins to degrade in a human or animal body1-3 years after implantation, or 2-5 years after implantation, or 3-10years after implantation, or longer.
 19. A composition comprising:ethylene vinyl alcohol (EVOH) dissolved in dimethyl sulfoxide (DMSO);wherein the EVOH comprises about 5-20 wt % of ethylene vinyl copolymer;wherein the ethylene vinyl copolymer comprises about 27-48 mole percentethylene; wherein the EVOH is capable of polymerizing into a polymercomprising a plurality of pores having an average pore size in the rangeof about 0.1 nm to about 3 μm.
 20. A method of occluding a body lumencomprising: imaging a body lumen using ultrasound; and percutaneouslyplacing a needle or catheter or portion thereof into the lumen;administering the composition of claim 1 into the lumen through theneedle or catheter; and allowing the composition to form an occlusion inthe lumen.